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Am J Pathol. May 2003; 162(5): 1693–1707.
PMCID: PMC1851199

Neuropathologies in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 Tat Protein under the Regulation of the Astrocyte-Specific Glial Fibrillary Acidic Protein Promoter and Doxycycline

Abstract

The human immunodeficiency virus type 1 (HIV-1) Tat protein is a key pathogenic factor in a variety of acquired immune deficiency syndrome (AIDS)-associated disorders. A number of studies have documented the neurotoxic property of Tat protein, and Tat has therefore been proposed to contribute to AIDS-associated neurological diseases. Nevertheless, the bulk of these studies are performed in in vitro neuronal cultures without taking into account the intricate cell-cell interaction in the brain, or by injection of recombinant Tat protein into the brain, which may cause secondary stress or damage to the brain. To gain a better understanding of the roles of Tat protein in HIV-1 neuropathogenesis, we attempted to establish a transgenic mouse model in which Tat expression was regulated by both the astrocyte-specific glial fibrillary acidic protein promoter and a doxycycline (Dox)-inducible promoter. In the present study, we characterized the phenotypic and neuropathogenic features of these mice. Both in vitro and in vivo assays confirmed that Tat expression occurred exclusively in astrocytes and was Dox-dependent. Tat expression in the brain caused failure to thrive, hunched gesture, tremor, ataxia, and slow cognitive and motor movement, seizures, and premature death. Neuropathologies of these mice were characterized by breakdown of cerebellum and cortex, brain edema, astrocytosis, degeneration of neuronal dendrites, neuronal apoptosis, and increased infiltration of activated monocytes and T lymphocytes. These results together demonstrate that Tat expression in the absence of HIV-1 infection is sufficient to cause neuropathologies similar to most of those noted in the brain of AIDS patients, and provide the first evidence in the context of a whole organism to support a critical role of Tat protein in HIV-1 neuropathogenesis. More importantly, our data suggest that the Dox inducible, brain-targeted Tat transgenic mice offer an in vivo model for delineating the molecular mechanisms of Tat neurotoxicity and for developing therapeutic strategies for treating HIV-associated neurological disorders.

Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an important role in the pathogenesis of a number of acquired immune deficiency syndrome (AIDS)-related disorders. 1,2 As one of the early HIV-1 proteins translated from the multiply spliced viral RNA transcripts, 3 Tat transactivates HIV-1 gene expression through interactions with the transactivation responsive element TAR within the HIV-1 long terminal repeat promoter, human cyclin T1, and CDK9. 4 This involves recruitment of an essential multicomponent factor, termed positive transcription elongation factor b (P-TEFb) to the HIV-1 long terminal repeat promoter, and phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). In addition to being a transactivator of HIV-1 gene expression, Tat has also been documented to exert pleiotropic effects on host cells, through direct modulation of gene expression by Tat uptake from extracellular microenvironment, and/or intracellular signaling elicited by interaction of extracellular Tat protein with cell surface receptors. 5-8 Corroborated with these functions, HIV-1 Tat has been demonstrated as secreted from Tat-expressing cells 9-11 and HIV-infected cells, 12,13 and as being capable of entering cells in a biologically active form. 14,15

HIV-1 infects the central nervous system (CNS) of a majority of AIDS patients, 16-18 and often leads to neurological symptoms such as memory loss and impaired cognitive and motor functions. 18,19 More than half of the pediatric AIDS patients, and ~20 to 25% of HIV-infected adults eventually develop dementia. 17,18,20,21 HIV-associated neuropathologies include reactive astrocytosis and cerebellar atrophy in the early stage of infection, and demyelination, formation of multinucleated giant cells, neuron death, and breakdown of the blood-brain barrier at later stages of the disease. 16,18,19,21-25 The target cells for HIV-1 infection in the brain are microglia/macrophages and astrocytes. 26,27 It has been generally accepted that neurons are most affected, although they are not directly infected. Thus, many indirect mechanisms have been explored and/or proposed for HIV-1 infection-induced neuropathogenesis. Those include HIV-1 viral proteins gp120 and Tat, and cellular factors secreted from HIV-infected macrophages/microglia and astrocytes, such as tumor necrosis factor-α, platelet-activating factor, arachidonic acid metabolites, oxygen-free radicals, nitric oxide, excitatory amino acids, and chemokines. 22,23,28-34 However, the precise in vivo role of these factors in contributing to HIV-associated CNS injury remains to be defined.

A number of studies have implicated Tat protein in HIV-induced neuropathogenesis. Tat is neurotoxic in vitro. 1,2 When injected into the brain, Tat protein causes histological changes similar to those seen in HIV-demented patients. 1,35 A recent study has suggested a positive correlation between the levels of Tat mRNA transcript and HIV- and simian-human immunodeficiency virus-induced encephalitis. 36 The mechanisms proposed for Tat neurotoxicity include direct depolarization of neurons, increased levels of intracellular calcium, increased production/release of proinflammatory cytokines, increased infiltration of macrophages/monocytes, activation of excitatory amino acid receptors, and increased apoptosis. 2 However, the argument has always been whether Tat is present within the CNS or cerebrospinal fluid in HIV-infected individuals at sufficient concentrations, to directly exhibit the acute neurotoxicity observed in most of those studies. Meanwhile, independent studies have shown that at lower concentrations Tat exhibits no acute neurotoxic activity, and instead transactivates gene expression and induces cell proliferation, differentiation, adhesion, and morphological changes. 7,37-39 In agreement with these observations, our recent studies suggest that Tat may affect neuronal function through direct uptake of extracellular Tat protein and subsequent disruption of neuronal metabolic balance of low-density lipoprotein receptor-related protein physiological ligands. 15 In addition, an overwhelming majority of the studies on Tat neurotoxicity has only focused on Tat interaction with neurons, and has not taken into account the interaction among neurons, astrocytes, and other brain cells. Nevertheless, it is certain that Tat interaction with astrocytes and other brain cells including endothelial cells also has adverse effects on neuronal survival and function. 34,40-43

Thus, it has become increasingly evident that the bona fide mechanisms of Tat neurotoxicity should be addressed in the context of a whole organism. There are several Tat transgenic mouse models available, 44-47 but none of them is suitable for studying Tat neurotoxicity. In those models Tat expression occurs constitutively throughout development and in all or most of tissues. As a result, any phenotypic abnormalities observed in the brain or a particular brain region could be caused by, or complicated by, abnormalities that occur in the brain any time during development or that exist in any other tissues or organs of the animal. In the present study we modified the doxycycline (Dox)-regulated gene expression strategy 48 with a brain-specific promoter, ie, the astrocyte-specific glial fibrillary acid protein (GFAP) promoter, 33,49,50 and generated the Dox-inducible and brain-targeted Tat transgenic mouse model. Characterization of phenotypes and neuropathologies of the transgenic mice concludes that Tat protein is a critical pathogenic factor in HIV-associated neuropathogenesis. More importantly, our data suggest that the unique Tat transgenic mouse model is very valuable for understanding the molecular mechanisms of Tat neurotoxicity, and for developing therapeutics for treating HIV-associated neurological diseases.

Materials and Methods

Plasmid Construction

The murine GFAP promoter in C-3123 plasmid (a gift from Dr. Lennart Mucke, University of California, San Francisco, CA 49 ) was released by restriction digestion with EcoRI and BamHI, and cloned into the pTeton (Clontech, Palo Alto, CA) in place of the cytomegalovirus promoter. The recombinant plasmid was designed as pTeton-GFAP. The standard polymerase chain reaction (PCR) cloning technique was used to construct pTRE-Tat86 plasmid. Briefly, HIV-1 Tat gene encoding 86 amino acids from both the first and second exons of HIV-1 isolate HXB2 was obtained by PCR using pBD-Tat86 15 as the template and primers 5′-GGA ATT CAC CAT GGA GCC AGT AGA TCC T-3′ (EcoRI site underlined) and 5′-CGG GAT CCC TAT TCC TTC GGG CCT GT-3′ (BamHI site underlined), and then cloned into EcoRI- and BamHI-digested pTRE vector backbone (Clontech). Similarly, we also constructed a control plasmid pTRE-CAT expressing the chloramphenicol acetyltransferase (CAT) reporter gene. All recombinant plasmids were verified by sequencing.

Cell Cultures, Transfection, and the CAT Assay

HeLa and U87.MG cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37°C with 5% CO2. HeLa (1 × 106 cells/100 mm plate) and U87.MG (0.5 × 106 cells/100 mm plate) cells were transfected with plasmid DNAs as indicated using the standard calcium phosphate method. Transfected cells were cultured in the presence of Dox (1 μg/ml) for 48 hours, and harvested for the CAT reporter gene assay using the phase extraction and direct scintillation counting method as previously described. 51 For U87.MG/iTat stable cells line, we first transfected U87.MG cells with the regulator plasmid pTeton-GFAP, and selected in the presence of 200 μg/ml of G418 for the stable transfectants, called U87.MG/GFAP cells. The stable transfectants were then functionally characterized by transient transfection of the expressor plasmid pTRE-CAT. The cell line with the highest transactivation of the CAT expression in the presence of Dox was selected and transfected with the expressor plasmid pTRE-Tat86. The selection was performed in the presence of 50 μg/ml of hygromycin (Life Technologies, Inc., Gaithersburg, MD) because a plasmid expressing the hygromycin-resistant gene (Clontech) was included in the transfection. The resulting stable cell line was designated U87.MG/iTat cells.

Creation of Inducible Tat Transgenic Mice

Inducible Tat transgenic mouse colonies (GT-tg) were obtained by generation of two separate transgenic lines Teton-GFAP mice (G-tg) and TRE-Tat86 mice (T-tg), and then cross-breeding of these two lines of transgenic mice. Briefly, a DNA fragment (2238 bp) containing the Teton-GFAP gene, along with downstream simian virus 40 splicing and polyadenylation sequences, was released by XhoI and PvuII digestion of the pTeton-GFAP plasmid and purified by agarose gel electrophoresis, and microinjected into fertilized eggs of F1 females obtained from mating between C3HeB and FeJ mice (Jackson Laboratories, Bar Harbor, ME). Founder transgenic animals were crossed with C57BL/6 mice to generate stable G-tg transgenic lines. Similarly, T-tg transgenic lines were obtained using a DNA fragment (1189 bp) released by XhoI and PvuII digestion of the pTRE-Tat86 plasmid. Founder animals and progeny carrying the transgenes were identified by PCR analysis of genomic DNA, which was extracted from mouse tail clippings (~0.5 to 1 cm long) using the Wizard genomic DNA isolation kit (Promega, Madison, WI). For amplification of the Teton-GFAP transgene, the primers 5′-GCT CCA CCC CCT CAG GCT ATT CAA-3′ and 5′-TAA AGG GCA AAA GTG AGT ATG GTG-3′ were used, whereas the TRE-Tat86 transgene was amplified with the primers 5′-CGG TGG GAG GCC TAT ATA AGC-3′ and 5′-AAC TGC AGT TAT TCC TTC GGG CCT GTC GG-3′. Fifty ng of genomic DNA was used in the PCR reactions, with a program of one cycle of 94°C for 3 minutes, 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute, and one cycle of 72°C for 7 minutes. The expected sizes of the amplified fragments for Teton-GFAP and TRE-Tat86 transgenes were 420 bp and 250 bp, respectively. Southern blot analysis was also performed to determine the presence and the copy number of the transgenes in mice using the 32P-labeled transgene probes.

Dox Treatment of Transgenic Mice

The animals were housed in the Laboratory Animal Care Center of Indiana University School of Medicine with a 12-hour light and 12-hour dark photoperiod. Water and food are provided ad libitum. All animal procedures were approved by the Institutional Biosafety Committee. In all experiments unless stated otherwise, comparisons were made between wild-type mice treated with Dox and Tat transgenic mice treated with and without Dox. Mice at postnatal day 21 (P21) were given Dox (Sigma, Louis, MO) in drinking water (in aluminum foil-wrapped bottles) at a concentration of 6 mg/ml for 7 days. Empty vehicle treatment was included as a control. Animals were sacrificed after the last day of Dox treatment, ie, P28, and the brains were removed and divided saggitally. One hemibrain was processed for RNA or protein isolation. The other hemibrain was fixed overnight in phosphate-buffered saline (PBS)-buffered 4% paraformaldehyde and processed for paraffin embedding.

RNA Isolation and Reverse Transcriptase (RT)-PCR Analysis of Tat Expression

Total RNA was isolated using the Trizol Reagents (Life Technologies, Inc.) according to the manufacturer’s instructions. Tat expression was analyzed using the Titan One Tube RT-PCR System kit (Boehringer Mannheim, Indianapolis, IN), with Tat-specific primers 5′-GGA ATT CAC CAT GGA GCC AGT AGA TCC T-3′ and 5′-CGG GAT CCC TAT TCC TTC GGG CCT GT-3′. RT-PCR was performed on a PE Thermocycler 9600 (PE Applied Biosystem, Foster City, CA) with a program of 50°C for 30 minutes, 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, and one cycle of 72°C for 7 minutes. Control RT-PCR reactions were performed in the absence of RT and genomic DNA templates to rule out the possibilities of genomic DNA contamination in RNA preparations and nonspecific amplification. In addition, the mouse glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was included in the RT-PCR as an internal control, with GAPDH-specific primers 5′-CTC AGT GTA GCC CAG GAT GC-3′ and 5′-ACC ACC ATG GAG AAG GCT GG-3′.

Immunohistochemistry

To ensure objective assessments and reliability of results, brain sections from mice to be compared in any given experiment were blind-coded and processed in parallels. Codes were revealed only after the analysis was complete.

Five-μm paraffin sections were cut on a microtome and mounted directly on glass slides for histochemical staining. The sections were deparaffinized in xylene and rehydrated, then stained using the Immunocruz Staining System (Santa Cruz Biotechnologies, Santa Cruz, CA) according to the manufacturer’s instruction. Anti-Tat antibody was a gift from Dr. Avindra Nath (University of Kentucky, Lexington, KY). All other antibodies used in our studies were from Santa Cruz Biotechnologies, including rabbit antibodies directed against GFAP (a marker for astrocytes) and CD14 (a marker for monocytes), and goat antibodies directed against MAP-2 (a marker for neurons) and CD68 (a marker for macrophages/microglia). Briefly, deparaffinized sections were unmasked by heating the sections at 95°C for 5 minutes in 10 mmol/L of sodium citrate buffer (pH 6.0) in a microwave with buffer exchanges for three times. After cooling to room temperature, the sections were pretreated in methanol containing 0.3% hydrogen peroxide (H2O2) for 30 minutes, blocked with 2.5% bovine serum albumin and 2% normal serum for 2 hours at room temperature, and then incubated with the appropriate primary antibodies at room temperature for 3.5 hours or at 4°C overnight, followed by an appropriate secondary biotinylated antibody for an additional 2 hours at room temperature. Bound antibodies were visualized with 3,3′-diaminobenzidine as the chromogen. All incubations were performed under humidified conditions, and slides were washed three times between steps with PBS. Finally, sections were counterstained in 1% hematoxylin. Omission of the primary antibodies in parallel stainings was included as a control. Stained sections were examined and bright-field microscopic images were captured with a Zeiss color camera mounted on a Zeiss Axiovert M200 fluorescence microscope using a ×20 plan apochromat objective, unless stated otherwise. A yellow background was generated by the microscope when the transmission light was selected to capture the immunohistochemical staining images. Image analysis was performed using an Axiovision 3.0 software package (Zeiss).

Cell Counting

The number of immunolabeled cells was counted manually on bright-field microscopic images in 1 of every 10 saggital sections (coded) by three independent individuals. All cell countings were location-matched between each group of mice. One frame for the visual field of 100 × 100 μm was used for cell counting. The analysis was performed on three animals of each group. Three different visual fields were chosen randomly within the same microanatomical brain regions of each animal. Average values of cell counts were calculated from the pooled data.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis for Tat and Interferon (IFN)-γ

Tat secretion in the supernatants of cell cultures and IFN-γ production in the brain homogenate were determined by Due Set ELISA Development System (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction. U87.MG/iTat stable cells were treated with or without 1 μg/ml of Dox, and the cell-free supernatants (0.2 ml) were collected for Tat determination at time points as indicated. For IFN-γ, the hemibrain was homogenized in 25 mmol/L of HEPES, pH 7.4, containing a cocktail of protease inhibitor called Complete TM (Boehringer Mannheim) on ice for 10 minutes. The homogenates were obtained by centrifugation at 4°C and 2000 × g for 5 minutes, followed by 13,000 × g for 30 minutes. The protein concentration was determined using a Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA). Serial dilutions of the supernatants or brain homogenates were performed to ensure the readings in the linear range. Recombinant Tat protein 15 and IFN-γ standards (R&D Systems) were used to calibrate the assay and determine the levels of Tat or IFN-γ. Antibodies for Tat and IFN-γ ELISA assay were from the National Institutes of Health AIDS Reagents Program and R&D Systems, respectively.

In Situ Apoptosis Detection

Five-μm paraffinized brain sections were deparaffinized and rehydrated as described above. Cell apoptosis was determined with the terminal deoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL)-based TdT-FragEL DNA Fragmentation Detection kit (Oncogene, Boston, MA). Briefly, deparaffinized sections were treated in 10 mmol/L of Tris-HCl, pH 8.0, containing 20 μg/ml of proteinase K at room temperature for 20 minutes, and in 3% H2O2 at room temperature for 5 minutes. The sections were then equilibrated with 1× TdT buffer, and incubated with TdT-labeling reaction mixture at 37°C for 2 hours. The labeling reaction was terminated by addition of the stop solution supplied in the kit, and incubation at room temperature for 5 minutes. The sections were rinsed with TBS (20 mmol/L Tris-HCl, pH 7.6, and 140 mmol/L NaCl) between steps. Finally, cell apoptosis was visualized with incubation of the sections in 3,3′-diaminobenzidine at room temperature for 15 minutes. Counterstain was performed in 0.3% methyl green solution after a thorough rinse with distilled water.

Statistical Analysis

Values were expressed as means ± SEM. Comparisons among groups were made using the Student’s t-test. A P value of <0.05 was considered statistically significant (*), and P < 0.001 highly significant (**).

Results

Astrocyte-Specific and Dox-Dependent Gene Expression in Vitro

Our goal was to determine the roles of Tat in HIV-1 neuropathogenesis. Because of possible acute neurotoxicity of Tat, we decided to use the tetracycline-regulated gene expression strategy, in which gene expression can be regulated by simple administration or withdrawal of tetracycline or its derivatives. This system consists of two components: 48 a tetracycline-responsive regulator (tTA) that is a fusion of Escherichia coli tet repressor to the transactivation domain of VP16 protein of herpes simplex virus under the cytomegalovirus (CMV) promoter, and a tTA-dependent, minimal RNA polymerase II promoter-driven expressor. Based on the relationship between tetracycline and gene expression, this system has evolved into two versions, ie, the Tet-off system in which addition of tetracycline prevents transcription by binding to tTA, and genes of interest are expressed only after tetracycline is removed, and the Tet-on system that uses a reverse tTA element (rtTA) and only becomes transcriptionally active when tetracycline or its derivatives such as Dox are present. A recent study has shown that a long-term administration of tetracycline has potential adverse effects on normal memory. 52 Thus, we chose the Tet-on system in our proposed studies. Moreover, to achieve brain-specific Tat expression, we decided to modify the Tet-on system by integrating into the regulator the astrocyte-specific GFAP promoter. Therefore, we made two transgenic constructs expressing each of these components. One was the regulator pTeton-GFAP, in which the CMV promoter was replaced by a 2.1-kb GFAP promoter 49 (Figure 1a) [triangle] . The other was the expressor pTRE-Tat86, in which the HIV-1 HXB2 Tat cDNA (86 amino acids in length) was cloned under the control of the tetO promoter (Figure 1a) [triangle] . Although Tat proteins from primary HIV-1 isolates have more than 86 amino acids, the 86-amino acid Tat gene that we used contains the most conserved domains of the Tat sequence of many HIV isolates (KT Jeang: HIV-1 Tat: structure and function. “The Human Retroviruses and AIDS Compendium on Line”: http://hiv-web.lanl.gov, 1994). First, we investigated the specificity of the GFAP promoter in the context of the pTeton backbone, ie, pTeton-GFAP. We transfected astroglial U87.MG cells and nonastroglial HeLa cells with pTRE-CAT expressing the CAT reporter gene, in combination with pTeton-GFAP, or pTeton (Figure 1a) [triangle] . pCMVβGal was included to normalize variations of transfection efficiency among transfections. As expected, in the absence of Dox treatment, transfection of pTRE-CAT with pTeton or pTeton-GFAP gave rise to little CAT activity in HeLa and U87.MG cells (Figure 1b) [triangle] . In the presence of Dox (1 μg/ml), co-transfection of pTRE-CAT with pTeton exhibited comparable levels of CAT expression between HeLa cells and U87.MG cells, which was 44.6 ± 3.8-fold and 40.4 ± 2.5-fold induction, respectively (Figure 1b) [triangle] . In contrast, in the presence of Dox treatment, co-transfection of pTRE-CAT with pTeton-GFAP transactivated CAT expression by 7.8 ± 0.7-fold in U87.MG cells, but only 1.4 ± 0.3-fold in HeLa cells (Figure 1b) [triangle] . These results confirmed that the GFAP promoter activity was astrocyte-specific. The inducibility of the CAT gene expression by the GFAP promoter, ie, sixfold to sevenfold was similar to the activity of the constitutive GFAP promoter. 53,54

Figure 1.
Transgenic constructs and Dox-dependent GFAP promoter activity. a: Transgenic constructs. pTRE-CAT was constructed as an expressor control for in vitro analysis. The GFAP promoter was cloned in place of the CMV promoter of pTeton regulator, while the ...

To further ensure the validity of the GFAP promoter-based Tet-on strategy for studying the effects of Tat in the CNS, we determined whether Tat was secreted out of astrocytes when expressed in astrocytes. Tat secretion as an extracellular protein is required for subsequent interaction of Tat with neurons, which mimics the brain of HIV-infected individuals. Thus, we stably transfected pTeton-GFAP and pTRE-Tat86 DNAs sequentially into U87.MG cells, and generated an inducible Tat astroglial cell line called U87.MG/iTat (see Material and Methods for details). We treated U87.MG/iTat cells with or without Dox, and determined levels of Tat protein in the culture supernatants. In the presence of Dox (1 μg/ml), Tat protein was detected in the supernatants at 12 hours after Dox treatment, and increased to a maximal level at 24 hours after Dox treatment (Figure 1c) [triangle] . In contrast, little Tat protein was detected in the supernatants of U87.MG/iTat cells in the absence of Dox treatment (Figure 1c) [triangle] . The concentrations of Tat protein in the supernatants were determined to be in the range of 100 to 850 pg/ml. These results confirmed that Tat was secreted once expressed in astrocytes. In addition, we did not detect cell death in Dox-treated U87.MG/iTat cells by the propidium iodide-staining method (data not shown), suggesting that Tat protein has no apparent adverse effects on U87.MG cells. These data together provided the initial evidence to support use of the modified Tet-on system to study Tat interaction with neurons in the transgenic mouse model.

Generation of the Dox-Regulated, Brain-Targeted Tat Transgenic Mouse Colonies

Next, we began to generate the Dox-regulated, brain-targeted Tat transgenic mice. Our strategy was to generate separate transgenic lines of mice containing the Teton-GFAP transgene (designated G-tg) or TRE-Tat86 transgene (designated T-tg), and then to crossbreed these two lines of transgenic mice to obtain the Dox-regulated, brain-targeted Tat bigenic mice (designated GT-tg; Figure 2a [triangle] ). Both Teton-GFAP and TRE-Tat86 transgenes were released by restriction digestion (XhoI and PvuII) from their respective plasmid DNAs pTeton-GFAP and pTRE-Tat86 (Figure 1a) [triangle] , purified, and microinjected into fertilized oocytes (C3HeB × FeJ). A total of five G-tg transgenic founders and seven T-tg transgenic founders were obtained. Crossing with the wild-type C57BL/6 mice was performed to stabilize each transgene. Then, G-tg transgenic mice were paired and crossed with T-tg transgenic mice to obtain GT-tg bigenic mice carrying both Teton-GFAP and TRE-Tat86 transgenes. The presence of transgenes was confirmed using genomic DNA PCR of the mouse tail clippings with transgene-specific primers (see Materials and Methods). Homozygous genotypes were confirmed by crossbreeding transgenic or bigenic mice with wild-type C57BL/6 mice. Representative genotyping results of transgenic and bigenic mice were shown in Figure 2b [triangle] . The copy numbers of transgenes were determined to be in the range of 3 to 10 (data not shown). A total of four GT-tg bigenic colonies was obtained. Although only GT-tg bigenic mice were needed for most of the studies (see below), we had also maintained the parental G-tg and T-tg transgenic lines. This would allow us to regenerate GT-tg bigenic mice in case the unexpected happens.

Figure 2.
Creation of Tat transgenic colonies. a: The creation scheme of the Dox-inducible and brain-targeted Tat transgenic mice. Teton-GFAP (G-tg) and TRE-Tat86 (T-tg) transgenic mice were generated independently, and then crossbred to obtain the Teton-GFAP and ...

Tat Expression in the Brain of Inducible Tat Bigenic Mice

Because there are no reliable anti-Tat antibodies available for Western blot analysis of native Tat protein, we decided to use RT-PCR, as well as in situ hybridization, immunohistochemical staining (see below), and ELISA analysis, to determine Tat expression in the brain of Tat transgenic mice. We first performed RT-PCR to determine whether Dox would induce Tat expression in the brain. We fed the GT-tg bigenic mice immediately after weaning, ie, 21 days of age, with Dox-containing (6 mg/ml) drinking water for 7 consecutive days, 55,56 and then harvested the brain for RNA isolation. The representative results showed that Dox treatment in the drinking water induced Tat mRNA expression in both heterozygous and homozygous GT-tg bigenic mice, and no Tat mRNA expression in the wild-type control mice (Figure 3a) [triangle] . To ensure no genomic DNA contamination in the RNAs isolated, RT-PCR reactions without addition of the RT were always performed as controls (data not shown). However, as reported elsewhere, 57 there was leaky expression of Tat mRNA transcripts in the GT-tg bigenic mice in the absence of Dox treatment (Figure 3a) [triangle] . We then determined whether Tat expression was regulated by Dox. We fed the GT-tg bigenic mice with the drinking water containing different concentrations of Dox. Consistent with previous studies, 55,56 the results showed that Tat expression began at 1.5 mg/ml of Dox, reached a plateau at 6 mg/ml of Dox, with no further increases when Dox concentration exceeded 6 mg/ml (Figure 3b) [triangle] . These results together showed that Tat expression in the brain of GT-tg bigenic mice was induced and regulated by Dox. Tat expression in the brain was also detected in supernatants of the primary astrocyte cultures prepared from the brain tissue of the GT-tg bigenic mice, as well as in the brain homogenates of the GT-tg bigenic mice using ELISA analysis (data not shown). In addition, there was a positive correlation between the copy numbers of the expressor TRE-Tat86 transgene and levels of Tat mRNA transcripts in GT-tg bigenic lines (data not shown). Nonetheless, the differences of Tat expression among the bigenic lines appeared well within the range of Tat expression at the concentrations of Dox examined (Figure 3b) [triangle] . Based on the maximal inducibility of Tat mRNA expression in the presence of Dox, these four GT-tg bigenic lines were designated as low-expressor line GT-tg181, intermediate-expressor lines GT-tg182 and GT-tg272, and high-expressor line GT-tg271. The high-expressor line GT-tg271 was used throughout our studies unless stated otherwise.

Figure 3.
Tat expression in the brain of the GT-tg bigenic mice. a: Dox-dependent Tat expression. GT-tg bigenic mice, 21 days old, were fed with Dox (6 mg/ml)-containing water for 7 consecutive days, and the brain was harvested. Age-matched wild-type mice were ...

We also determined whether the modified Tet-on strategy would specifically target Tat expression within the brain. We fed the GT-tg bigenic mice with the Dox-containing water for 7 days, and harvested various organs of the GT-tg bigenic mice, such as eye, heart, kidney, liver, lung, spleen, and thymus. We performed RT-PCR for Tat expression in those tissues. The results showed that Tat expression occurred in the brain, but not in other tissues or organs, after the GT-tg bigenic mice were systematically exposed to Dox (Figure 3c) [triangle] . The amplification products of genomic DNA in these nonbrain tissues or organs was confirmed to be nonspecific by Southern blot analysis using a TRE-Tat transgene-specific DNA probe (data not shown). Moreover, consistent with the astroglial GFAP expression pattern, 58,59 in situ hybridization detected higher levels of Tat mRNA in the cerebellum, cerebellar cortex, hippocampus, brain stem, tectum, and white matter tracts in the brains of GT-tg bigenic mice treated with Dox, much fewer Tat mRNA transcripts in the brains of GT-tg bigenic mice treated without Dox, and no Tat mRNA transcripts in the brains of wild-type mice (data not shown).

Developmental and Behavioral Abnormalities of Tat-Expressing Mice

To assess the effects of Tat expression on mouse development, we fed the mice at 21 days of age with Dox (6 mg/ml)-containing water and recorded the changes of the body weight. Before initiation of Dox treatment, GT-tg bigenic mice appeared to be normal, and indistinguishable from the age-matched wild-type mice, G-tg transgenic, or T-tg transgenic mice. When treated with Dox, the GT-tg bigenic mice exhibited significant growth failure (Figure 4a) [triangle] , which has also been reported in the mice expressing the NL4-3 Δgag/pol transgene. 60,61 By the end of the 7-day Dox treatment (6 mg/ml), the wild-type mice gained 4.51 ± 0.26 g (n = 8, P < 0.001), the G-tg transgenic mice for 4.32 ± 0.21 g (n = 6, P < 0.001), and the T-tg transgenic mice for 4.47 ± 0.25 g (n = 8, P < 0.001), whereas the GT-tg bigenic mice showed little growth (0.66 ± 0.21 g, n = 20, the bigenic line GT-tg271, P > 0.05) (Figure 4b) [triangle] . To ascertain that the growth failure was because of Tat expression, we treated the GT-tg bigenic mice with different concentrations of Dox (Figure 4c) [triangle] . In agreement with induction of Tat expression (Figure 3b) [triangle] , the least growth was found in mice fed with 6 mg/ml of Dox (0.76 ± 0.27 g, n = 5, P > 0.05), followed by 3 mg/ml of Dox (1.81 ± 0.28 g, n = 6; P < 0.001), 1.5 mg/ml Dox (3.45 ± 0.24 grams, n = 5; P < 0.001), and no Dox treatment (3.27 ± 0.19 g, n = 6; P < 0.001), whereas the wild-type mice gained 4.21 ± 0.23 g (n = 4, P < 0.001) when treated with Dox at the highest concentration, ie, 6 mg/ml. Similar patterns of growth failure were obtained in other three bigenic lines GT-tg181, GT-tg182, and GT-tg272 (data not shown). These results together demonstrated that growth retardation was because of induction of Tat gene expression in the brain, other than an insertional mutation with neurodevelopmental consequences, or other unknown nonspecific effects.

Figure 4.
Tat expression-induced growth failure. a: Appearance of the GT-tg bigenic mice treated with or without Dox. b: Effects of Tat expression on body growth of mice. GT-tg bigenic mice, 21 days of age (filled bars), were fed with Dox-containing water (6 mg/ml) ...

In addition to growth failure, we also noted a number of behavioral changes and/or neurological signs inTat-expressing bigenic mice. Those changes included hunched posture, tremor, ataxia, hindlimb weakness, slow motor and cognitive movement, and seizures (Table 1) [triangle] . By week 1 to week 2 after Dox treatment, affected mice were 16 of 16 in the bigenic line GT-tg181, 9 of 10 in the bigenic line GT-tg182, 17 of 22 in the bigenic line GT-tg271, and 12 of 15 in the bigenic line GT-tg272. In the absence of Dox, bigenic lines exhibited similar phenotypic features, but to a much lesser extent, ie, 6 of 14 in the bigenic line GT-tg181, 2 of 8 in the bigenic line GT-tg182, 6 of 15 in the bigenic line GT-tg271, and 4 of 12 in bigenic line GT-tg272. In addition, some of the affected mice died at an early age (3 to weeks), typically during and perhaps as a result of seizures. The death rate was 5 of 17 of affected mice in the bigenic line GT-tg271, 3 of 12 of affected mice in the bigenic line GT-tg272, and 1 of 6 of affected mice in the bigenic line GT-tg271. The severity of the abnormal changes and death rate appeared to be correlated with the levels of Tat expression in the brain. Interestingly, the GT-tg bigenic mice that survived the first 2 weeks after Dox treatment showed some improvement in body growth for several weeks, but progressively developed tremor, ataxia, and seizures, and eventually died by 3 months of age.

Table 1.
Tat Expression and Abnormalities in Mice

Tat Expression-Induced Neuropathologies

We then characterized the neuropathologies of Tat-expressing mice. We first performed hematoxylin and eosin staining of the brain sections. Histological examination revealed complete collapse of the cerebellum, progressive loss of the cortex, and significant vacuolation in the brain of the GT-tg bigenic mice treated with Dox, and the dead GT-tg bigenic mice from Dox treatment (Figure 5) [triangle] . The cerebellum was severely afflicted in the brain of the GT-tg bigenic mice treated with Dox, or the dead GT-tg bigenic mice from Dox treatment and as result, became disintegrated during the tissue processing (Figure 5, C and D) [triangle] . In addition, Tat expression was associated with increased hematoxylin and eosin staining in the nuclei, suggesting that Tat expression resulted in cell death, mostly neurons determined by cell morphology. Although inflammation (encephalitis or meningitis) was only occasionally observed, hydroencephalitis or edema was often present in the GT-tg bigenic mice when treated with Dox.

Figure 5.
Disruption of cerebella and cortex and neuronal death in the brain of Tat-expressing mice. H&E staining was performed in the sections of brain from wild-type mice treated with Dox (6 mg/ml) for 7 days, GT-tg bigenic mice treated with or without ...

Because astrocytosis is a hallmark of HIV-induced neuropathologies, 62-64 we next determined whether Tat expression in the astrocytes would induce astrocytosis. We performed immunohistochemical staining of brain sections using anti-GFAP antibody, and anti-Tat antibody (a gift from Dr. A. Nath, University of Kentucky, Lexington, KY). We also included a normal rabbit IgG as a staining control (data not shown). Consistent with levels of Tat expression, the astrocytes in the brain of the Dox-treated GT-tg bigenic mice had a morphology typical of reactive astrocytosis, 65 showing enlarged cell bodies and an increased GFAP immunoreactivity (Figure 6) [triangle] . The GT-tg bigenic mice also showed some morphological changes of astrocytes in the absence of Dox treatment and perhaps the GFAP immunoreactivity, but to a much lesser extent. Meanwhile, the age-matched, wild-type control mice showed a normal distribution and morphology of GFAP-positive astrocytes.

Figure 6.
Astrocytosis, loss of neuron dendrite, and neuronal apoptosis in the brain of Tat-expressing mice. The serial sections of the brain of the wild-type mice treated with Dox and GT-tg bigenic mice treated with or without Dox were immunolabeled with anti-Tat ...

We then evaluated the integrity of neurons in the brain of Tat-expressing mice. We performed immunohistochemical staining of brain sections using an antibody against a dendrite marker, microtubule-associated protein 2 (MAP2). Surprisingly, digital-scanning microscopic imaging showed significant neuronal and dendritic changes in cerebellar, cortical, and hippocampal neurons. Compared to the age-matched, wild-type normal mice, the GT-tg bigenic mice exhibited considerable loss of dendritic processes, and atrophic and tortuous neuronal body (Figure 6) [triangle] . The terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining further confirmed a significant increase in positively stained neurons (apoptotic neurons) in the brain of the Dox-treated GT-tg bigenic mice (Figure 6) [triangle] . The apoptotic neurons appeared to be in a close proximity to Tat-positive regions including cerebellum, cortex, and hippocampus.

Tat Expression and the CNS Infiltration

Macrophage/monocyte infiltration into the CNS plays an important role in AIDS-associated dementia. HIV-1 Tat protein is chemotactic by itself, or through up-regulation of chemokine and chemokine receptor expression. Thus, we next examined the relationship between Tat expression and macrophage/monocyte and lymphocyte infiltration. We performed immunohistochemical staining of the brain sections with antibodies against CD14 (monocyte marker) and CD68 (macrophage/microglia marker), as well as antibodies against CD4 (CD4 T lymphocytes) and CD8 (CD8 T lymphocytes). Examination of stained brain sections revealed that there were considerable increases in CD14-, CD4-, and CD8-positive cells, but fewer changes in CD68-positive cells in the brain of the GT-tg bigenic mice treated with Dox (Figure 7a) [triangle] . There also appeared even more CD14-, CD4-, and CD8-positive cells in the brain of the dead GT-tg bigenic mice from Dox treatment. Moreover, the CD4- and CD8-positive cells in the brain of the dead GT-tg bigenic mice exhibited a morphology typical of T lymphocytes in an activated stage (Figure 7a) [triangle] . Quantitative analysis of these brain sections showed that the number of CD4-positive cells was 165.2 ± 24.1 in the wild-type mice and 135.8 ± 15.2 in the GT-tg bigenic mice in the absence of Dox treatment, which was increased to 250.6 ± 34.4 in the GT-tg bigenic mice when treated with Dox, and 481.5 ± 40.2 in the dead Dox-treated GT-tg bigenic mice. Meanwhile, the number of CD8-positive cells was 15.3 ± 3.2 in the wild-type mice and 35.4 ± 4.6 in the GT-tg bigenic mice in the absence of Dox treatment, which was increased to 141.2 ± 12.7 in the GT-tg bigenic mice when treated with Dox, and 253.3 ± 17.8 in the dead Dox-treated GT-tg bigenic mice. Similarly, the number of CD14-positive cells also showed considerable increases, ie, 35.6 ± 5.2 in the wild-type mice, 60.1 ± 4.8 in the GT-tg bigenic mice treated without Dox, 85.6 ± 5.9 in the GT-tg bigenic mice treated with Dox, and 161.4 ± 14.3 in the dead Dox-treated GT-tg bigenic mice. Nevertheless, no marked changes were noted in the number of CD68-positive cells, ie, 65.3 ± 6.3 in the wild-type mice, 67. 3 ± 4.8 in the GT-tg bigenic mice treated without Dox, 70.6 ± 3.4 in the Dox-treated GT-tg bigenic mice, and 68.4 ± 6.2 in the dead Dox-treated GT-tg bigenic mice (Figure 7b) [triangle] .

Figure 7.
The CNS infiltration induced by Tat expression. a: Increased staining of CD14-, CD4-, and CD8-positive, but not CD68-positive cells in the brain of Tat-expressing mice. The serial sections of the brain of the wild-type mice treated with Dox, the GT-tg ...

T lymphocytes normally enter the CNS only after they are activated by contact with helper cells and other antigens. 66,67 The CNS infiltration of activated CD4 and CD8 cells has been demonstrated in viral infection-induced neurological disorders. 68,69 Thus, we next determined whether T lymphocytes infiltrated into the CNS of the Tat-expressing mice were activated. We prepared brain homogenates and measured production of IFN-γ, a molecular marker for CD8 cell activation. Consistent with the morphological changes (Figure 7a) [triangle] , the results showed that IFN-γ production was markedly increased from 184.6 ± 78.8 pg/mg protein in the Dox-treated wild-type mice and 140.9 ± 18.2 pg/mg protein in the GT-tg bigenic mice treated without Dox to 939.8 ± 185.9 pg/mg protein in the Dox-treated GT-tg bigenic mice (Figure 7C) [triangle] , confirming that those infiltrated T lymphocytes were activated.

Discussion

In this study, we demonstrated that brain-targeted expression of HIV-1 Tat protein was sufficient to cause a number of developmental and behavioral abnormalities including failure to thrive, hunched posture, tremor, ataxia, slow motor and cognitive movement, seizures, and premature death, and extensive neuropathological changes such as loss of cerebellum and cortex, neuronal death (apoptosis), astrocytosis, degeneration of neuronal dendrites, and the CNS infiltration of monocytes and activated T lymphocytes. The phenotypic and neuropathological features noted in the GT-tg bigenic mice correlated with the level and spatial distribution of Tat mRNA and/or protein expression and occurred in four different GT-tg bigenic lines when treated with Dox but not in these bigenic mice treated without Dox, or in the Dox-treated wild-type normal mice. Therefore, all of these findings did not result from an insertional mutation, or other unknown nonspecific effects, and support the notion that HIV-1 Tat protein has direct and wide-ranging pathogenic actions in the CNS and has an important role in HIV-associated neuropathogenesis.

Unlike most of the published studies, we attempted to address Tat neurotoxicity in the context of a whole organism. To this end, we integrated the astrocyte-specific GFAP promoter into the inducible Tet-on gene expression system. We chose the GFAP promoter to target astrocytes as Tat-expressing cells in the CNS for the following reasons: 1) Tat has direct and acute neurotoxic activity, which may not allow Tat interaction with neurons as an extracellular (soluble) factor if Tat is expressed in neurons; 2) there are no microglia/macrophage-specific promoters available, although HIV-1 primarily infects microglia/macrophages in the CNS; 3) Tat has been found to have no apparent cytotoxicity on astrocytes; 4) increasing evidence suggests that astrocytes are involved in the pathogenesis of HIV-associated neurological diseases; 23,70-73 5) the GFAP promoter-driven gene expression in astrocytes has been successfully used to characterize the CNS functions of a number of genes including HIV-1 gp120, lipoprotein E4 (ApoE4), and cytokines such as interleukin (IL)-3, IL-6, IL-12, tumor necrosis factor-α, and IFN-α. 53 . Our results confirmed that the astrocyte-specific GFAP promoter effectively targeted Tat expression in astrocytes, and in a Dox-dependent manner both in vivo and in vitro (Figures 1 and 3) [triangle] . Nevertheless, consistent with an earlier report, 57 our results indicated that this modified system exhibited a minimal level (leaky) of Tat expression in the absence of Dox (Figure 3) [triangle] . However, our results showed that the inducibility of Tat expression by Dox treatment fell within the range of Tat expression among the GT-tg bigenic lines containing different copies of the transgene TRE-Tat86 (data not shown), which may be because of the inherited lower activity of the GFAP promoter. 33,50,53,54 Interestingly, it has been recently demonstrated that Dox has significant protective effects on neurons against apoptosis induction and global ischemia. 74,75 Therefore, the actual adverse effects of Tat on the development, behavior, and neuropathology of the GT-tg mice would likely be much greater.

Our results showed that Tat expression induced growth failure, and other apparent abnormalities such as hunched posture, tremor, ataxia, and slow motor and cognitive movement, seizures, and subsequent premature death (Figure 4 [triangle] and Table 1 [triangle] ). Similar results have been reported in several HIV-1 transgenic studies, 60,61,76 as well as HIV-infected children and adults. 77-80 Although the mechanisms are currently unknown, a multifactorial etiology seems likely. At the cellular level, Tat expression in the brain of the GT-tg bigenic mice resulted in degeneration of cerebellum and cortex, and neuronal death (Figure 5) [triangle] . Compared to mild cerebellar atrophy often identified in the brain of HIV-infected individuals, 81-84 Tat-expressing mice exhibited complete collapse of the cerebellum, which may likely result from rapid induction and localized accumulation of Tat protein in the cerebellum within such a short period of time, ie, 7 days, because less severe cerebellar damage was evident in the brain of GT-tg bigenic mice treated with Dox at the concentrations of 1.5 and 3 mg/ml (data not shown). Moreover, induction of astrocytosis, loss of neuron dendrites, and neuron apoptosis were also noted in the brains of Tat-expressing mice (Figure 6) [triangle] . Similar neuropathologies have been described in the brain of HIV-infected individuals. 35, 62-64, 85 Consistent with the results obtained from a rat model, 35 our data demonstrated that Tat increased the CNS infiltration of monocytes and T lymphocytes (Figure 7, a and b) [triangle] , which we further confirmed was accompanied by increased production of potential neurotoxic molecules such as IFN-γ (Figure 7c) [triangle] . This activity is apparently because of Tat expression and secretion, and/or Tat-mediated modulation of chemokines/chemokine receptors and cytokines in the CNS.

Although the exact molecular basis for the neurodegenerative changes seen in the brain of Tat-expressing GT-tg bigenic mice, including the unusual diffuse nuclear and perinuclear TUNEL-staining pattern in the brain of Tat-expressing mice, is under investigation in the laboratory, it is undoubtedly clear that the changes are a combined result of Tat expression in astrocytes, Tat interactions with neurons, other brain cells, including microglia and brain capillary endothelial cells, and infiltrated monocytes and T lymphocytes, and the interactions among those cells. More importantly, the unique transgenic mouse model will help to define the molecular mechanisms of Tat neurotoxicity, and develop and evaluate novel therapeutics for treating HIV-associated neuropathologies.

Acknowledgments

We thank Dr. Lennart Mucke for the GFAP promoter plasmid, Dr. Avindra Nath for anti-Tat antibodies, and Dr. Loren Field for technical assistance on the transgenic work.

Footnotes

Address reprint requests to Johnny J. He, Department of Microbiology and Immunology, Indiana University School of Medicine, R4 302, 1044 W. Walnut St., Indianapolis, IN 46202. E-mail: .ude.iupui@ehjj

Supported by the National Institutes of Health (grants R01NS39804 and R01MH65158 to J. J. H.) and the Ralph W. and Grace M. Showalter Trust Foundation (an award to J. J. H.).

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