Axonal transport of human immunodeficiency virus type 1 envelope protein glycoprotein 120 is found in association with neuronal apoptosis

Abstract

Patients infected by human immunodeficiency virus type 1 (HIV-1) develop acquired immune deficiency syndrome-associated dementia complex (ADC), a disorder characterized by a broad spectrum of motor impairments and cognitive deficits. The number of cells in the brain that are productively infected by HIV-1 is relatively small and consists predominantly of macrophages and microglia, yet HIV-1 causes widespread neuronal loss. A better understanding of the pathogenic mechanisms mediating HIV-1 neurotoxicity is crucial for developing effective neuroprotective therapies against ADC. The HIV-1 envelope glycoprotein 120 (gp120), which is shed from the virus, is one of the agents causing neuronal cell death. However, the cellular mechanisms underlying its neurotoxic effect remain unclear. We report that gp120 injected into the rat striatum or hippocampus is sequestered by neurons and subsequently retrogradely transported to distal neurons that project to these brain areas. Cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, hallmarks of apoptosis, were seen in neurons internalizing and transporting gp120. The retrograde transport of gp120 and apoptosis were mediated by the chemokine receptor CXCR4 because AMD3100, a selective CXCR4 inhibitor, blocked both events. Furthermore, colchicine or nocodazole, two inhibitors of intracellular trafficking, abolished gp120-mediated apoptosis in distal areas. These results indicate that axonal transport of gp120 might play a role in HIV-1-mediated widespread neuronal cell death.

Figure 1.

Neuronal internalization of gp120. a, VEH or gp120IIIB was injected into the striatum (see boxed area) as described previously (Nosheny et al., 2004). Animals were killed 24 h later. gp120-IR was examined by immunohistochemistry in serial sections from the striatum. b–d, Higher magnifications of boxed area in a. Sections were stained with a gp120 antibody (green), followed by NeuN (b, red), GFAP (c, red), or CD68 (d, red). Yellow is green plus red. Note that gp120-IR in d follows the needle track. Scale bar, 100 μm.

Figure 2.

AMD3100 reduces apoptosis. Rats received VEH (1), AMD3100 (2), gp120 alone (3) or in combination with AMD3100 (4) into the striatum and were killed 24 h (a, b) or 4 d (c, d) later. a, b, Representative striatal sections of rats treated with gp120 incubated overnight with an antibody against gp120 (green) along with a specific CXCR4 antibody (red). Yellow is red plus green. Scale bars: a, 30 μm; b, 50 μm. The number of cells positive for gp120-IR (c) and caspase-3-IR (d) were counted in sections throughout the striatum in an area 250 μm². Data, expressed as the mean ± SEM of four animals (at least 10 sections both caudal and rostral to the injection site per animal), represent the average number of positive cells per section. ∗p < 0.01 versus gp120.

Figure 3.

Retrograde axonal transport of gp120. Rats received gp120 into the striatum and were killed at different days after the injection. a, b, Representative sections from the SN of gp120-treated rats (killed 4 d after the injection) stained with TH (red) and gp120 (green) antibodies. b, Higher magnification of the boxed area in a. Yellow is green plus red. c, d, Representative sections from the somatosensory cortex of animals treated with gp120, stained with neurofilament (red) and gp120 (green) antibodies. d, Higher magnification of boxed area in c. gp120-IR is present mainly in neurofilament-positive cells in layer V. Yellow is green plus red). Scale bars: a, 60 μm; b, 50 μm; c, 100 μm; d, 30 μm. e, Time course analysis of gp120 accumulation in the SN and cerebral cortex. Rats received gp120 into the striatum and were killed at 1, 2, 4, 7, and 15 d after the injection. Analysis of gp120-IR was performed in the indicated areas in a total of 15 sections per animal (8 rats each time point), one every 100 μm. Data are the mean ± SEM.

Figure 4.

Hippocampal gp120 is retrogradely transported to distal neurons. Rats received gp120 (400 ng) into the dorsal hippocampus or fimbria and were killed 1, 2, 4, 7, and 15 d later. a–c, Sections throughout the ipsilateral hippocampus of gp120-treated rats (killed 1 d after the injection into the hippocampus), contralateral hippocampus (d) (4 d after the injection), medial septum (e), and diagonal band of Broca (f) (4 d after the injection into the fimbria), were stained for gp120 (green), NeuN (red, a, e, f), GFAP (red, b), MAP2 (red, d), and CXCR4 (red, c). Yellow is red plus green. Scale bars: a, c, f, 125 μm; b, d, e, 100 μm. g, gp120-positive neurons were quantified in the septum and contralateral hippocampus at the indicated days after the injection. Data, expressed as the mean ± SEM of eight animals (13 and 16 sections through the septum and hippocampus each, respectively, 1 every 100 μm, per animal), represent the average number of cells per section.

Figure 5.

Neurons transporting gp120 are TUNEL and caspase-3 positive. Rats received gp120 (400 ng) into the striatum or hippocampus and were killed at 1, 2, 4, 7, and 15 d after the injection. Representative sections from the SN (a) or contralateral hippocampus (b) taken from animals 7 d after the injection, stained with cleaved caspase-3 (red) and gp120 (green) antibodies. a and b show that gp120-positive neurons in the SN and contralateral hippocampus are also caspase-3 positive. Scale bars: a, 30 μm; b, 50 μm. c, d, Time course analysis of gp120-IR colocalization in TUNEL- or caspase-3-positive cells, respectively, in the indicated regions. Data represent the mean ± SEM of eight rats per group (at least 10 sections each area, each animal).

Figure 6.

Colchicine blocks gp120 retrograde transport and apoptosis. Rats received VEH (1), gp120 (2), colchicine (3), or colchicine plus gp120 (4) into the striatum and were killed 3 d later. Representative sections from the SN of rats treated with gp120 (a) or gp120 plus colchicine (b) showing colocalization of gp120-IR (green) in TH (red)-positive neurons. Note that colocalization occurs only in a. Scale bar, 50 μm. Semiquantitative analysis of TH- and gp120-positive neurons (c) and caspase-3- and TH-IR (d) in sections from the SN. Number of gp120-IR neurons (e) and caspase-3-positive neurons (f) in the striatum. Data, expressed as the mean ± SEM of four animals per group (at least 10 sections per animal), represent the average number of positive cells per section. ∗p < 0.001 versus gp120.

Figure 7.

AMD3100 blocks gp120-mediated activation of caspase-3 in the SN. Rats received VEH (1), gp120 (2), AMD3100 (3), or AMD3100 plus gp120 (4) into the striatum and were killed 4 d later. Semiquantitative analysis of TH/gp120 (a) and TH/caspase-3-positive neurons (b) in the SN. Data, expressed as the mean ± SEM of four animals per group (at least 10 sections per animal), represent the average number of positive cells per section. ∗p < 0.001 versus gp120.

Figure 8.

Subcellular localization of gold-labeled gp120 in cerebellar granule cells. Representative electron microscopic images of CGCs taken at 5 (A), 15 (B), 30 (C), and 60 (D) min after gp120 incubation. The gold-labeled gp120 (arrows) is first seen attached to apparent receptor sites outside synaptic endings (A) and after internalization into synaptic terminals (B). At 30 min (C), the gold-labeled gp120 can be seen in association with microtubules within neuronal processes and at 60 min (D) within lysosomes in the cell body. Scale bars: A, B, D, 500 nm; C, 200 nm.

Figure 9.

Nocodazole blocks gp120-mediated axonal transport and retraction. CGCs were exposed to gp120 (5 nm) for 30 min (a), nocodazole (5 μg/ml) plus gp120 for 30 min (b), gp120 for 6 h (c), or nocodazole plus gp120 for 6 h (d). Nocodazole was added to the cultures 15 min before gp120. Neurons were fixed and stained for gp120 (green), DAPI (blue), or MAP2 (red). Note in a gp120-IR in neuronal processes (arrows). Scale bar, 20 μm. e, Caspase-3-positive neurons were determined 30 min and 6 h after gp120 and nocodozole (noc) alone or in combination. Values are the mean ± SEM (n = 8). ∗p < 0.001 versus control; ^#p < 0.005 versus gp120.