HIV gp120 upregulates tonic inhibition through α5-containing GABAARs

Abstract

HIV-Associated Neurocognitive disorder (HAND) affects nearly half of infected patients. The HIV envelope protein gp120 is shed by infected cells and is a potent neurotoxin in vitro that reproduces many aspects of HAND when expressed in vivo. Here, we show that HIV gp120 increases the amplitude of a tonic current mediated by γ-aminobutyric acid type-A receptors (GABA_ARs). Treating rat hippocampal cultures with 600 pM gp120_IIIB for 4 h increased a tonic bicuculline-sensitive current, which remained elevated for 24 h. The increased current resulted from upregulation of extrasynaptic α5-containing GABA_ARs, as indicated by inhibition with the selective inverse agonist basmisanil. Treatment with gp120 increased α5-GABA_AR immunoreactivity on the cell surface without new protein synthesis. The increase in tonic inhibition was prevented by a C-X-C chemokine receptor type 4 (CXCR4) antagonist or elimination of microglia from the culture. Treatment with interleukin-1β (IL-1β) increased the tonic current and an IL-1 receptor antagonist blocked the gp120-evoked response. Pharmacological or genetic inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the gp120-evoked increase in tonic current and direct activation of a mutant form of p38 MAPK expressed in neurons increased the current. Collectively, these data show that gp120 activates CXCR4 to stimulate microglia to release IL-1β. Subsequent stimulation of IL-1 receptors activates p38 MAPK in neurons leading to the upregulation of α5-containing GABA_ARs. Increased tonic inhibition impairs neuroplasticity and inhibition of α5-containing GABA_ARs improves cognitive function in disease models. Thus, gp120-induced upregulation of α5-containing GABA_ARs presents a novel therapeutic target for HAND.

Keywords: Basmisanil; Basmisanil, 1,1-Dioxidothiomorpholino) (6-((3-(4-fluorophenyl)-5-methylisoxazol-4-yl)methoxy)pyridin-3-yl)methanone (PubChem CID: 57336276); HAND; HIV gp120; α5 GABA(A)R subunit.

Figure 1:. gp120 increases tonic GABA_AR current.

(A) Representative traces show gp120 treatment (600 pM for 4 or 24 h) increases the amplitude of the bicuculline-sensitive shift in holding current compared to untreated neurons (scale bar: 50 pA, 10 s). (B) Summary plot showing effect of gp120 on bicuculline-sensitive currents normalized to whole cell capacitance (One-way ANOVA; F_(2,18)=6.21, p=0.009). Data are expressed as individual data points with mean ± SEM. One-way ANOVA was followed with Tukey’s post hoc test, *p<0.05 compared to untreated group.

Figure 2:. Upregulation of tonic inhibition is mediated by α5-containing GABA_ARs.

(A) Representative traces show currents sensitive to basmisanil, an α5-containing GABA_AR inverse agonist (10 nM) in untreated and gp120-treated cells (scale bar: 40 pA, 10 s). (B) Basimisanil-sensitive currents normalized to whole-cell capacitance from gp120-treated and untreated cells are compared (Unpaired two-tailed t-test; t₍₁₀₎=3.77, p=0.004). (C) Representative traces show gp120 did not change the amplitude of currents evoked by the agonist for δ-containing GABA_ARs, THIP (1 µM), compared to untreated controls (scale bar: 10 pA, 10 s). (D) THIP evoked current densities were similar in control and gp120-treated cells (Unpaired two-tailed t-test; t₍₁₀₎=0.12, p=0.9). (E) Representative images show surface expression of α5-immunoreactivity increased in gp120 treated relative to untreated cells. (F) The average immunofluorescence intensity from the soma of gp120-treated cells increased relative to untreated somata (Unpaired two-tailed t-test; t₍₅₂₎=3.417, p=0.001). Data are shown as individual data points with mean ± SEM (B,D) or expressed as mean ± SEM (F). ^***p<0.001 compared to untreated group.

Figure 3:. gp120 activates CXCR4 to stimulate microglia and increase tonic inhibition.

(A) The CXCR4 antagonist, AMD3100 (1 µM), inhibits gp120-induced (600 pM) increases in bicuculline-sensitive holding current normalized to whole-cell capacitance (two-way ANOVA; F_(1,28)=6.00, p=0.02). (B) Representative images showing microglia labeled by Ox-42 antibody (yellow) were depleted relative to MAP2 immunoreactive neurons (red) 24 h after treatment with LME (25 mM for 1 h) compared to DMEM wash controls. (C) Removal of microglia with LME blocks gp120-induced increases in tonic current (two-way ANOVA; F_(1,21)=7.63, p=0.01) compared to DMEM wash. Data are expressed as individual data points with mean ± SEM. Two-way ANOVAs were followed by Tukey’s post hoc test, ^**p<0.01 compared to untreated or DMEM control. ^##p<0.01 compared to gp120 alone (untreated or DMEM).

Figure 4:. gp120-induced release of IL-1β increases tonic inhibition.

(A) Representative traces show IL-1β (20 ng/mL for 20 min) alone increases the bicuculline sensitive current (scale bar: 50 pA, 30 s). (B) IL-1β significantly increases bicuculline-sensitive currents normalized to whole-cell capacitance compared to untreated cells (Mann-Whitney U-test; p=0.002). (C) Representative traces show that treatment with IL-1ra (1 µg/mL) blocks the gp120-induced increase in bicuculline-sensitive current. (D) IL-1ra significantly blocks the gp120-induced increase in bicuculline-sensitive current normalized to whole cell capacitance (two-way ANOVA; F _(1,19)=8.19, p=0.01). Data are expressed as individual data points with mean ± SEM. Two-way ANOVA was followed by Tukey’s post hoc test, *p<0.05, ^**p<0.01 compared to untreated control. ^#p<0.05 compared to gp120 alone (untreated).

Figure 5:. Neuronal p38 MAPK activation is required for gp120-induced increases in tonic inhibition.

(A) The p38 antagonist SB203,580 (10 µM) inhibits gp120-induced increases in bicuculline-sensitive current normalized to whole-cell capacitance (two-way ANOVA; F _(1,21)=9.14, p=0.006). (B) SB202,474 (10 µM), an inactive analog of SB203580, does not affect the gp120-induced increase in bicuculline-sensitive current (two-way ANOVA; F_(1,17)=1.09, p=0.3). (C) RapRp38 expression prevents the gp120-induced increase in tonic inhibition (two-way ANOVA; F_(1,27)=5.02, p=0.03). (D) Treatment with rapamycin (200 nM) increases tonic inhibition in RapRp38 expressing cells significantly more than cells expressing tdTomato alone (two-way ANOVA; F_(1,22)=6.08, p=0.02). (E) Cycloheximide (CHX) (10 µM) does not affect the gp120-induced increase in tonic inhibition (two-way ANOVA, F_(1,24)=0.003, p=0.9). Data are expressed as individual data points with mean ± SEM. Two-way ANOVAs were followed by Tukey’s post hoc test, *p<0.05, ^**p<0.01, ^***p<.001 compared to untreated control. ^# p<0.05, ##p<0.01, compared to gp120 (A,C) or rapamycin (D) alone (untreated or TdTomato).

Figure 6:. Hypothetical signaling pathway: HIV gp120 activates microglial and neuronal signaling pathways to upregulate α5-containing GABA_ARs.

Inhibition of CXCR4 with AMD3100 prevents gp120-induced changes in GABA_AR signaling. Because gp120_IIIB binds to CXCR4 (Islam et al., 2013; Moore et al., 1997) we propose that the signaling cascade starts with this receptor. While multiple cell types including neurons, astrocytes, and microglia express CXCR4, LME treatment to remove microglia prevented gp120-induced changes in GABA_AR signaling in neurons. Thus, gp120 does not act directly on neurons to evoke the inhibitory current and microglia are necessary and upstream from the neuronal response. Additionally, because IL-1ra blocked the gp120-induced increase in tonic inhibition and microglia release IL-1β in response to gp120 (Lisi et al., 2011), we placed microglia upstream from the neuron. Finally, p38 signaling in neurons is necessary for the gp120-induced increases in tonic inhibition as shown by RapRp38 expression in neurons. P38 activation is a canonical downstream effector of the IL-1R (Weber et al., 2010), consistent with IL1Rs on neurons mediating the response. P38 activation in other cell types may contribute to upstream steps in the pathways such as IL-1β release from microglia (Schieven, 2005), although these roles were not tested in this study. Activation of p38 increased α5-containing GABA_ARs on the neuronal cell surface without the need for protein synthesis; the final steps through which p38 regulates GABA_AR trafficking are not known. Selective inhibitors are shown in red.