HIV-1 Tat Induces Unfolded Protein Response and Endoplasmic Reticulum Stress in Astrocytes and Causes Neurotoxicity through Glial Fibrillary Acidic Protein (GFAP) Activation and Aggregation

Abstract

HIV-1 Tat is a major culprit for HIV/neuroAIDS. One of the consistent hallmarks of HIV/neuroAIDS is reactive astrocytes or astrocytosis, characterized by increased cytoplasmic accumulation of the intermediate filament glial fibrillary acidic protein (GFAP). We have shown that that Tat induces GFAP expression in astrocytes and that GFAP activation is indispensable for astrocyte-mediated Tat neurotoxicity. However, the underlying molecular mechanisms are not known. In this study, we showed that Tat expression or GFAP expression led to formation of GFAP aggregates and induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in astrocytes. In addition, we demonstrated that GFAP up-regulation and aggregation in astrocytes were necessary but also sufficient for UPR/ER stress induction in Tat-expressing astrocytes and for astrocyte-mediated Tat neurotoxicity. Importantly, we demonstrated that inhibition of Tat- or GFAP-induced UPR/ER stress by the chemical chaperone 4-phenylbutyrate significantly alleviated astrocyte-mediated Tat neurotoxicity in vitro and in the brain of Tat-expressing mice. Taken together, these results show that HIV-1 Tat expression leads to UPR/ER stress in astrocytes, which in turn contributes to astrocyte-mediated Tat neurotoxicity, and raise the possibility of developing HIV/neuroAIDS therapeutics targeted at UPR/ER stress.

Keywords: Glial fibrillary acidic protein; HIV-1 Tat; astrocytes; endoplasmic reticulum stress; endoplasmic reticulum stress (ER stress); glial cell; human immunodeficiency virus (HIV); neuron; unfolded protein response; unfolded protein response (UPR).

FIGURE 1.

Tat expression increased eIF-2α phosphorylation and Xbp-1 alternate splicing in astrocytes. A, iTat primary astrocytes were cultured in the presence of 5 μg/ml Dox for 0 and 3 days. Total RNA was isolated for RT-PCR using Tat- and GFAP-specific primers. GAPDH was included as an internal control. B, cell lysates of the same cells were separated into Triton X-100-soluble (TS) and insoluble (TIS) fractions and analyzed for GFAP expression by Western blotting. β-Actin was included as an equal loading control. C, the same cells were immunostained for GFAP expression, followed by microscopic imaging. The cells were counterstained with 100 ng/ml DAPI for nuclei. D and E, iTat primary astrocytes were cultured in the presence of 5 μg/ml Dox for 0, 1, 2, and 3 days. Cells were analyzed for total eIF-2α, phosphorylated eIF-2α (p-eIF-2α), or GFAP expression by Western blotting (D). β-Actin was included as an equal loading control. Total RNA was also isolated from the cells for RT-PCR using Xbp1- or Tat-specific primers (E). -Fold changes in protein expression over day 0 were the mean ± S.D. (error bars) of three independent repeats and are shown to the right of the respective Western blots. GAPDH was included as an internal control. U and S, unspliced and spliced RNA-derived PCR DNA, respectively. *, p < 0.05.

FIGURE 2.

Tat expression increased BiP expression in the mouse brain. Mice (3 mice/strain) were injected i.p. with Dox (80 mg/kg/day, + Dox) or pH-matched PBS (pH 2.2, −Dox) for 7 days. The brains were collected and sectioned, and comparable cortical sections were stained with anti-GFAP or anti-BiP antibody, followed by secondary antibody goat anti-rabbit Alexa Fluor 488 or goat anti-mouse Alexa Fluor 555. The sections were then counterstained with 100 ng/ml DAPI for nuclei. The comparable cortical regions of the brains were imaged. A total of three brain sections from three Dox-treated and three PBS-treated mice were stained. The images were representative of all of the stained cortical sections in each strain (A). -Fold changes in protein expression over the control (−Dox) were the mean ± S.D. (error bars) of multiple cortical sections from each strain and are shown beside the respective images of each group (B). *, p < 0.05.

FIGURE 3.

Ectopic GFAP expression activated ER stress in astrocytes. Mouse primary astrocytes (A and B) or human primary fetal astrocytes (C) were transfected with cDNA3 (C3), Tat·Myc, GFAP, or GFAP mutant 239H expression plasmids. The cells were analyzed for GFAP, Tat (c-Myc), BiP, total eIF-2α, phospho-eIF-2α (p-eIF-2α), ATF6, or OASIS expression by Western blotting at 3 days post-transfection (A and C), or the mouse astrocytes were analyzed at 1, 2, or 3 days post-transfection for Xbp-1 alternate splicing by RT-PCR (B). Treatment of mouse astrocytes with thapsigargin (500 nm) was included as a positive control (+) and PBS was included as a negative control (−) for Xbp-1 alternate splicing. U and S, unspliced and spliced RNA-derived PCR DNA, respectively. D, -fold changes in protein expression over the control (C3) were the mean ± S.D. (error bars) of three independent repeats. Open bars, data from A; closed bars, data from C. E, total RNA was isolated from C3- and Tat-transfected mouse primary astrocytes at day 3 and analyzed for GFAP, BiP, ATF6, and OASIS mRNA expression using quantitative RT-PCR. β-Actin was included as an internal control. -Fold changes in mRNA expression over the control (C3) were the mean ± S.D. of three independent repeats. *, p < 0.05.

FIGURE 4.

GFAP knock-out abolished Tat-induced astrocyte-mediated Tat neurotoxicity in vitro and ER stress in the brain. A, primary astrocytes were isolated from WT, iTat, or iTat/GFAP knock-out (iTat/GFAP−) mice and cultured in the presence of 5 μg/ml Dox for the indicated days, and the culture supernatants were collected and assayed for their neurotoxicity in SH-SY5Y. The data are the mean ± S.D. (error bars) of triplicates. B–E, WT, iTat, or iTat/GFAP− mice (3 mice/strain) were injected i.p. with Dox (80 mg/kg/day) for 0 and 7 days. The brains were collected and sectioned, and comparable cortical sections were stained with an antibody for BiP (B) or MAP-2 (C), followed by goat anti-mouse Alexa Fluor 488. -Fold changes in BiP expression (D) or MAP-2 expression (E) over the control (WT at day 0) were the mean ± S.D. of multiple cortical sections from each strain. *, p < 0.05.

FIGURE 5.

GFAP knock-out abolished Tat-induced ER stress in astrocytes in vitro. Primary astrocytes were isolated from iTat and iTat/GFAP− mice; cultured in the presence of 5 μg/ml Dox for 0, 1, 2, and 3 days; and then analyzed for GFAP, BiP, total eIF-2α, phospho-eIF2α (p-eIF-2α), ATF6, and OASIS expression by Western blotting (A). -Fold changes in protein expression over day 0 were the mean ± S.D. (error bars) of three independent repeats and are shown to the right of the respective Western blots. Total RNA was isolated from those cells for Xbp1 alternative splicing and Tat expression by semiquantitative RT-PCR (B). GADPH was included as a control. U and S, unspliced and spliced RNA-derived PCR DNA, respectively. Total RNA was also analyzed for GFAP, BiP, ATF6, and OASIS mRNA expression by quantitative RT-PCR (C). β-Actin was included as an internal control. -Fold changes in mRNA expression over the control (day 0) were the mean ± S.D. of three independent repeats. *, p < 0.05.

FIGURE 6.

4-PBA inhibited Tat-induced ER stress in astrocytes and astrocyte-mediated Tat neurotoxicity. A and B, U373.MG cells were transfected with cDNA3, Tat, or GFAP expression plasmid; cultured for 48 h; and continued to culture in the presence of 0 and 5 mm 4-PBA for 24 h. A fraction of the cells were harvested and analyzed for BiP expression by Western blotting, and total RNA was isolated from the remaining cells for Xbp-1 alternate splicing by semiquantitative RT-PCR (A). The culture supernatants from the 4-PBA-treated cells were evaluated for neurotoxicity using the MTT assay (B). C and D, iTat primary astrocytes were cultured in the presence of 5 μg/ml Dox for 0 and 3 days and then in the presence of 0, 0.1, 1, 5, or 10 mm 4-PBA for 24 h. A fraction of the cells were harvested and analyzed for BiP expression by Western blotting, and total RNA was isolated from the remaining cells for Xbp-1 alternate splicing by semiquantitative RT-PCR (C). U and S, unspliced and spliced RNA-derived PCR DNA, respectively. The culture supernatants from the 4-PBA-treated cells were evaluated for neurotoxicity using the MTT assay (D). -Fold changes in BiP expression over the control (C3 at day 0) were the mean ± S.D. (error bars) of three independent repeats and are shown below the respective Western blots (A and C). *, p < 0.05.

FIGURE 7.

4-PBA inhibited GFAP activation and formation of shortened neuron dendrites in the brain of iTat mice. WT and iTat mice (3 mice/strain) were injected i.p. with Dox (80 mg/kg/day) for 4 days and then with Dox (80 mg/kg/day) and 4-PBA (120 mg/kg/day) for an additional 3 days. The brains were collected 1 day after the final injection and sectioned, and comparable cortical sections were stained with an antibody for GFAP or BiP, followed by goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555, respectively. The sections were also counterstained with 100 ng/ml DAPI for nuclei. The images are representative of all of the stained cortical sections from each strain (A). -Fold changes in protein expression over the control (WT, −4-PBA) were the mean ± S.D. (error bars) of multiple cortical sections from each strain (B). *, p < 0.05.