HIV-1 Tat Promotes Lysosomal Exocytosis in Astrocytes and Contributes to Astrocyte-mediated Tat Neurotoxicity

Abstract

Tat interaction with astrocytes has been shown to be important for Tat neurotoxicity and HIV/neuroAIDS. We have recently shown that Tat expression leads to increased glial fibrillary acidic protein (GFAP) expression and aggregation and activation of unfolded protein response/endoplasmic reticulum (ER) stress in astrocytes and causes neurotoxicity. However, the exact molecular mechanism of astrocyte-mediated Tat neurotoxicity is not defined. In this study, we showed that neurotoxic factors other than Tat protein itself were present in the supernatant of Tat-expressing astrocytes. Two-dimensional gel electrophoresis and mass spectrometry revealed significantly elevated lysosomal hydrolytic enzymes and plasma membrane-associated proteins in the supernatant of Tat-expressing astrocytes. We confirmed that Tat expression and infection of pseudotyped HIV.GFP led to increased lysosomal exocytosis from mouse astrocytes and human astrocytes. We found that Tat-induced lysosomal exocytosis was tightly coupled to astrocyte-mediated Tat neurotoxicity. In addition, we demonstrated that Tat-induced lysosomal exocytosis was astrocyte-specific and required GFAP expression and was mediated by ER stress. Taken together, these results show for the first time that Tat promotes lysosomal exocytosis in astrocytes and causes neurotoxicity through GFAP activation and ER stress induction in astrocytes and suggest a common cascade through which aberrant astrocytosis/GFAP up-regulation potentiates neurotoxicity and contributes to neurodegenerative diseases.

Keywords: astrocyte; cathepsin B (CTSB); exocytosis; glial cell; human immunodeficiency virus (HIV); neuron.

FIGURE 1.

Neurotoxicity of Tat-immunodepleted supernatants from Tat-expressing astrocytes. U373.MG cells were transfected with pcDNA3 or pTat.Myc. Culture supernatants were harvested 72 h post-transfection and incubated with anti-Myc antibody for Tat immunodepletion (ID/α-Myc). Normal rabbit IgG was used as the control (ID/IgG). In addition, the supernatant without immunodepletion (No ID) was also included as a control. All supernatants were exposed to LTR-Luc-transfected 293T for their activity on the LTR promoter (A) or exposed to SH-SY5Y and evaluated for their effects on cell viability (B). Error bars, S.D.; *, p < 0.05.

FIGURE 2.

Relative contribution of several known factors to astrocyte-mediated Tat neurotoxicity. Wild-type primary astrocytes (WT) or iTat primary astrocytes were induced with Dox for 3 days. Supernatants were collected and incubated with PBS, WP9QY (5 and 10 μm), l-NAME (10 and 20 μm), SOD (10 and 20 μg/ml), or MK-801 (0.5 and 2 μm) or heat-inactivated. After the indicated treatment, supernatants were added into mouse primary neurons, cultured for 72 h, and evaluated for neurotoxicity using the MTT assay. WT supernatants were similarly treated and showed no neurotoxicity (data not shown). All comparisons were made against iTat treated with PBS. Error bars, S.D.; *, p < 0.05.

FIGURE 3.

Tat expression induced lysosomal exocytosis in astrocytes. A, iTat primary astrocytes were cultured in the absence (Dox−) or in the presence (Dox+) of 5 μg/ml Dox for 3 days. The cells were then processed for the NAG assay. The data are the mean ± S.D. of triplicate samples and are representative of three independent experiments. B and C, WT or iTat primary astrocytes were cultured in the presence of 5 μg/ml Dox for 3 days and then processed for TIRF. Each exocytosis event was counted manually (B, bottom panel and insets) based on the drastic drop of the fluorescence intensity within seconds shown in the histogram (B, top). One hundred quinacrine-labeled ATP-containing vesicles were randomly selected in each astrocyte. Three astrocytes from each sample were randomly selected for quantitation of lysosomal exocytosis events. The number of lysosomal exocytosis events is expressed as a percentage of the total number of vesicles (C). Error bars, S.D.; *, p < 0.05.

FIGURE 4.

Lysosomal exocytosis from Tat-expressing astrocytes and VSV-G-pseudotyped HIV.GFP-infected astrocytes. U373.MG cells were transfected with pcDNA3 (C3) or Tat.Myc (A) or infected with VSC-G-pseudotyped HIV.GFP (B), or human primary fetal astrocytes were infected with VSC-G-pseudotyped HIV.GFP (C). The cells were analyzed for NAG release 72 h post-transfection or 48 h post-infection using the NAG assay. Error bars, S.D.; *, p < 0.05.

FIGURE 5.

Lysosomal exocytosis from Tat-expressing astrocytes using cell surface LAMP-1 as a marker. WT and iTat primary astrocytes were cultured in the presence of 5 μg/ml Dox for 3 days, processed for cell surface LAMP-1 staining, and evaluated by confocal imaging for LAMP-1 staining (A) and by flow cytometry for quantitation of LAMP-1+ cell percentage (B). A and B, representative of three independent experiments; C, mean ± S.D. of LAMP-1+ cell percentages from three independent experiments. Error bars, S.D.; *, p < 0.05.

FIGURE 6.

Inhibition of lysosomal exocytosis decreased astrocyte-mediated Tat neurotoxicity. iTat primary astrocytes were cultured in the absence or presence of 5 μg/ml Dox for 3 days and then in the presence of the indicated concentrations of vacuolin-1 for 1 h. The cells were then processed for the NAG assay (A). The culture supernatants from the vacuolin-1-treated cells were collected and used to determine the neurotoxicity in SH-SY5Y cells using the MTT assay (B). Error bars, S.D.; *, p < 0.05.

FIGURE 7.

Cathepsin B inhibitor inhibited the neurotoxicity of the supernatants of Tat-expressing astrocytes. A, human primary fetal astrocytes were transfected with pcDNA3 (C3) or Tat, cultured for 72 h, and then treated with cathepsin B inhibitor Z-FA-fmk (100 μm) for 24 h. DMSO, the solvent of Z-FA-fmk, was included as a control. The supernatants were collected and used to determine the neurotoxicity in SH-SY5Y using the MTT assay. B–D, primary astrocytes were isolated from WT and iTat mice and cultured in the presence of Dox for 3 days and treated with cathepsin B inhibitor Z-FA-fmk as above. The supernatants were evaluated for their neurotoxicity as above (B). The cells were harvested for cell lysates. The cathepsin B (Cath. B) in both supernatants and cell lysates was determined by Western blotting (C). In addition, the supernatants were incubated with different concentrations of an anti-cathepsin B antibody (Cath. B) for 1 h and assayed for their neurotoxicity as above (D). Error bars, S.D.; *, p < 0.05.

FIGURE 8.

Effects of Tat expression on lysosome exocytosis and neurotoxicity in 239T and Huh 7.5.1. 293T (A) and Huh 7.5.1 cells (B) were transfected with cDNA3 (C3) or Tat.Myc. The cells were analyzed for the NAG release using the NAG assay. The supernatants were collected for their neurotoxicity in human primary fetal neurons (C) and SH-SY5Y (D) using the MTT assay. Error bars, S.D.; *, p < 0.05.

FIGURE 9.

GFAP knock-out abrogated Tat-induced lysosomal exocytosis in Tat-expressing astrocytes. Primary astrocytes were isolated from iTat and iTat/GFAP− mice and cultured in the absence or presence of 5 μg/ml Dox for 3 days. The cells were processed for the NAG assay (A). The supernatants were analyzed for their neurotoxicity using the MTT assay (B). Error bars, S.D.; *, p < 0.05.

FIGURE 10.

4-PBA inhibited lysosomal exocytosis in astrocytes. A, U373.MG were transfected with cDNA3, Tat, or GFAP expression plasmid, cultured for 48 h, and continued to culture in the absence or presence of 5 mm 4-PBA for 24 h. The cells were harvested and analyzed for the NAG release using the NAG assay. B, 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. The cells were harvested and analyzed for the NAG release using the NAG assay. Error bars, S.D.; *, p < 0.05.

FIGURE 11.

Working model for astrocyte-mediated Tat neurotoxicity. Tat is secreted from HIV-infected macrophages/microglia (1); Tat is taken up into astrocytes or expressed in HIV-infected astrocytes (2); Tat activates GFAP expression (3); increased GFAP expression leads to formation of GFAP aggregation (4); GFAP aggregation triggers UPR/ER stress (5); ER stress leads to calcium release into the cytoplasm (6); increased intracellular calcium stimulates Ca²⁺-dependent lysosomal exocytosis (7) and release of lysosomal hydrolytic enzymes and cathepsins into the extracellular space (8); and lysosomal hydrolytic enzymes and cathepsins cause neurotoxicity (9).