Activation of p38 MAPK Is Required in Monocytic and Neuronal Cells for HIV Glycoprotein 120-Induced Neurotoxicity

Abstract

HIV-1 envelope protein gp120 has been implicated in neurotoxin production by monocytic cells, namely macrophages and microglia, and the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). We previously showed in cerebrocortical cell cultures from rodents containing microglia, astrocytes and neurons, that overall inhibition of p38 MAPK signaling abrogated the neurotoxic effect of HIV-1 gp120. However, the time course of p38 MAPK activation and the contribution of this kinase in the various cell types remained unknown. In this study, we found that for HIV gp120-induced neurotoxicity to occur, active p38 MAPK is required in monocytic lineage cells, namely macrophages and microglia, and neuronal cells. In cerebrocortical cell cultures HIV-1 gp120 stimulated a time-dependent overall increase of active p38 MAPK and the activated kinase was primarily detected in microglia and neurons. Interestingly, both increased activation of p38 MAPK and neuronal death in response to gp120 were prevented by prior depletion of microglia, or in the presence of CCR5 ligand CCL4 or of p38 MAPK inhibitors. In human monocytic THP-1 cells and primary monocyte-derived macrophages (MDM), HIV gp120 stimulated production of neurotoxins was abrogated by prior introduction into the cells of a dominant-negative p38 MAPK mutant or p38 MAPK siRNA. In addition, the neurotoxic effects of cell-free supernatants from gp120-stimulated monocytic THP-1 cells were prevented in microglia-depleted cerebrocortical cells pretreated with a pharmacological inhibitor of p38 MAPK. Thus, p38 MAPK signaling was critical upon exposure to HIV gp120 for both the neurotoxic phenotype of monocytic cells and subsequent toxin-initiated neuronal apoptosis.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage

“…Rat mixed neuronal-glial cerebrocortical cell cultures were prepared from embryos of Sprague-Dawley rats at days 15 to 17 of gestation, as previously described by our group (44)(45)(46). In brief, cells were cultured in 35-mm dishes with poly-L-lysine-coated glass coverslips (1.87 ϫ 10 6 cells per dish) or in poly-L-lysine-coated clear-bottom 96-well plates for imaging (0.087 ϫ 10 6 cells per well) (BD Falcon, BD Biosciences, San Jose, CA).…”

“…Cells were cultured in D10C medium containing 70% Dulbecco's modified Eagle's medium (DMEM) with high glucose (Gibco, Life Technologies), 12.5% heat-inactivated horse serum (Gibco, Life Technologies), 12.5% F-12 medium (HyClone, Thermo Scientific), 3.12% 1 M HEPES (Omega Scientific), 1.25% 200 mM L-glutamine (Gibco, Life Technologies), and 0.3% 100 U/ml penicillin with 100 mg/ml streptomycin (Gibco, Life Technologies). Cell cultures consisting of ϳ30% neurons, ϳ70% astrocytes, and ϳ0.1 to 1% microglia were used after 16 days in vitro, when the majority of neurons were considered fully differentiated and susceptible to NMDA toxicity (45 Immunocytochemistry/immunofluorescence staining and microscopy. After treatment and washing with phosphate-buffered saline (PBS), rat cerebrocortical cell cultures were fixed for 25 min with 4% paraformaldehyde (PFA) at 4°C and subsequently permeabilized with 0.2% Triton X-100 for 5 min at room temperature.…”

“…Microscopy was performed using filters for 4=,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC), and Cy3, using either an Axiovert 100M or 200M microscope (Zeiss). Images were acquired and fluorescence intensities for MAP-2, synaptophysin, and nuclear DNA were quantified using the Slidebook software package, versions 4 and 5 (Intelligent Imaging Innovations, Denver, CO), as described earlier (44,45,47). The two microscope setups used were both controlled by Slidebook software and equipped with the same objectives but had different light sources and cameras.…”

“…The cell lysates were transferred to microcentrifuge tubes, sonicated four times for 5 s each time, and then cleared by centrifugation (13,200 rpm, 10 min) at 4°C. Total protein concentrations in lysates were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Thermo Fisher Scientific, Rockford, IL) (45).…”

HIV Toxins: Gp120 as an Independent Modulator of Cell Function

“…In other study, Gp120-mediated neurotoxicity was found to involve signaling trough the p38 MAPK in macrophages, microglia and neuronal cells. Gp120-mediated p38 MAPK activation and neuronal death was prevented by CCL4 (MIP-1beta), one of the CCR5 ligands (Medders et al, 2010). On the other hand, soluble Tat is able to cross the BBB and to induce the production of chemoattractive factors by astrocytes and monocytes (mainly MCP-1, which is considered one of the most important chemokines in HIV infection and HAND), and the expression of CCR5 on monocytes (Weiss et al, 1999).…”