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HIV-1 gp120 Promotes Lysosomal Exocytosis in Human Schwann Cells

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HIV-1 gp120 Promotes Lysosomal Exocytosis in Human Schwann Cells

Gaurav Datta et al. Front Cell Neurosci.

Abstract

Human immunodeficiency virus type 1 (HIV-1) associated neuropathy is the most common neurological complication of HIV-1, with debilitating pain affecting the quality of life. HIV-1 gp120 plays an important role in the pathogenesis of HIV neuropathy via direct neurotoxic effects or indirect pro-inflammatory responses. Studies have shown that gp120-induced release of mediators from Schwann cells induce CCR5-dependent DRG neurotoxicity, however, CCR5 antagonists failed to improve pain in HIV- infected individuals. Thus, there is an urgent need for a better understanding of neuropathic pain pathogenesis and developing effective therapeutic strategies. Because lysosomal exocytosis in Schwann cells is an indispensable process for regulating myelination and demyelination, we determined the extent to which gp120 affected lysosomal exocytosis in human Schwann cells. We demonstrated that gp120 promoted the movement of lysosomes toward plasma membranes, induced lysosomal exocytosis, and increased the release of ATP into the extracellular media. Mechanistically, we demonstrated lysosome de-acidification, and activation of P2X4 and VNUT to underlie gp120-induced lysosome exocytosis. Functionally, we demonstrated that gp120-induced lysosome exocytosis and release of ATP from Schwann cells leads to increases in intracellular calcium and generation of cytosolic reactive oxygen species in DRG neurons. Our results suggest that gp120-induced lysosome exocytosis and release of ATP from Schwann cells and DRG neurons contribute to the pathogenesis of HIV-1 associated neuropathy.

Keywords: ATP; DRG neuron; P2X4; Schwann cell; gp120; lysosome exocytosis.

Figures

FIGURE 1
FIGURE 1
HIV-1 gp120 induces lysosomal exocytosis in primary human Schwann cells (hSCs). (A) Compared to heat inactivated gp120 (control), gp120 caused a concentration-dependent increase in the activity of acid phosphatase in media of hSCs (n = 5, *p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). (B) HIV-1 gp120 did not change release of LDH in hSCs. (C) gp120 treatment (8.3 nmol/L for 40 min) significantly increased the release of acid phosphatase in the media of hSCs (n = 3, *p < 0.05). (D) In surface protein biotinylation assay, gp120 treatment (8.3 nmol/L for 40 min) increased PM levels of LAMP1 (n = 3, ∗∗∗p < 0.001). (E) In surface protein labeling assay, gp120 treatment (8.3 nmol/L for 40 min) increased PM of LAMP1 (n = 3, ∗∗p < 0.01; bar = 15 μm).
FIGURE 2
FIGURE 2
HIV-1 gp120 causes lysosomal redistribution in hSCs. (A) Classification scheme of LAMP1 positive lysosomes in concentric shells labeled as peripheral (0–2 μm from PM), juxtanuclear (2–4 μm from PM), and perinuclear (>4 μm from PM) lysosomes. (B) As shown in representative LAMP1 staining (insert) and 3D reconstructed images, gp120 treatment (8.3 nmol/L for 40 min) increased the percentage of peripheral (green) and juxtanuclear (magenta) lysosomes and decreased the percentage of perinuclear (yellow) lysosomes. Plasma membranes were outlined with Pan-Cadherin (red) and the nucleus stained with DAPI (blue). (C) gp120 treatment (8.3 nmol/L for 40 min) did not change total numbers of LAMP1 positive lysosomes (n = 100) (D) gp120 treatment (8.3 nmol/L for 40 min) did not change TFEB protein levels in hSCs.
FIGURE 3
FIGURE 3
HIV-1 gp120-induces redistribution of lysosomes displays functional heterogeneity. (A) gp120 treatment (8.3 nmol/L for 40 min) significantly decreased colocalization of LAMP1 (green) with RILP (red) in peripheral lysosomes, but increased colocalization of LAMP1 with RILP in juxtanuclear lysosomes (n = 3, *p < 0.05, ∗∗∗p < 0.001). (B) As shown in representative confocal (inset) and reconstructed Imaris images, gp120 treatment (8.3 nmol/L for 40 min) significantly reduced Cathepsin B activity (red) in peripheral (white) lysosomes (n = 3, ∗∗∗p < 0.001).
FIGURE 4
FIGURE 4
Lysosomal P2X4 channel is involved in HIV-1 gp120 induced lysosomal exocytosis. (A) Representative confocal and reconstructed Imaris images show P2X4 (red) is colocalized with LAMP1 (green) positive lysosomes in hSCs (bar = 20 μm). (B) In surface biotinylation assay, gp120 treatment (8.3 nmol/L for 40 min) increased P2X4 translocation to the PM in hSCs (n = 3, ∗∗∗p < 0.001). (C) An allosteric P2X4 regulator Bx430 (0.5 μmol/L) prevented gp120 (8.3 nmol/L for 40 min)-induced increases in the activity of acid phosphatase release in media of hSCs (n = 3, *p < 0.05, ∗∗p < 0.01). (D) As shown in RT4 rat Schwann cells co-transfection of P2X4-pHluorin123 (GFP) and LAMP1-RFP (bar = 10 μm), gp120 treatment (8.3 nmol/L for 40 min) increased fluorescence of P2X4-pHluorin123 (n = 3, ∗∗∗p < 0.001).
FIGURE 5
FIGURE 5
Lysosomal ATP transporter-VNUT mediates HIV-1 gp120 induced lysosomal exocytosis. (A) Representative confocal images and scatterplot show the colocalization of VNUT (green) with LAMP1 (red) positive lysosomes in hSCs (bar = 15μm). (B) Inhibiting VNUT with clodronate (Clo, 0.1 μmol/L) prevented gp120 (8.3 nmol/L for 40 min)-induced increases in the activity of acid phosphatase release in media of hSCs (n = 3, ∗∗p < 0.01). (C) gp120 treatment (8.3 nmol/L for 40 min) increased ATP levels in media of hSCs, and this effect was prevented by inhibiting VNUT with clodronate (Clo, 0.1 μmol/L) (n = 3, ∗∗p < 0.01. (D) As shown in representative Imaris reconstructed and confocal images (inset), gp120 (8.3 nmol/L for 40 min) increased percentage of ATP (BODIPY FL-ATP, green) in peripheral and juxtanuclear lysosomes (LAMP1-RFP) (n = 3, ∗∗p < 0.01).
FIGURE 6
FIGURE 6
HIV-1 gp120 conditioned RT4 Rat Schwann cell media increases cellular calcium and ROS in DRG neurons. (A) gp120 conditioned Schwann cell media, but not gp120 + Bx430 or gp120 + Clo conditioned media, increased intracellular calcium levels as measured by Fluo-8 in rat DRG neurons (n = 3, ∗∗p < 0.01). (B) gp120 conditioned media, but not gp120 + Bx430 or gp120 + Clo conditioned media, increased cytosolic ROS levels as measured by DCF-DA in rat DRG neurons (n = 3, ∗∗p < 0.01).

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