gp120-induced neurotoxicity in hippocampal pyramidal neuron cultures: protective action of TGF-beta1

Abstract

We found that TGF-beta1, a cytokine that previously has been reported to have neuroprotective effects, was able to prevent the toxicity induced by the HIV-1 coat protein gp120 in hippocampal pyramidal neuron cultures. In the presence of glia, gp120 induced time- and dose-dependent cell death, which was more pronounced in mature (7-19 d in culture) than in young neurons (2-7 d in culture). Staining with nuclear dyes (propidium iodide and Hoechst 33342), in situ detection of DNA fragments, and DNA analysis on agarose gels indicated that apoptosis was mainly responsible for the death caused by the viral protein. However, after several days of treatment, death-displaying necrotic features also occurred. Neurotoxicity induced by gp120 was dependent on the activation of NMDA receptors and required the presence of glia as well as new protein synthesis. Thus, the effect of gp120 was abolished by the NMDA receptor antagonist APV and partially reduced by cycloheximide. Only modest neurotoxicity was observed in pure neuronal cultures deprived of the glia feeder layer. Fura-2-based videoimaging showed that treatment with gp120 enhanced the ability of NMDA to increase neuronal [Ca2+]i. The impairment of neuronal Ca2+ homeostasis was prevented completely by TGF-beta1. Therefore, it is likely that the neuroprotective action of the cytokine is attributable to its ability to stabilize neuronal [Ca2+]i.

Fig. 1.

Effect of recombinant gp120 on the survival of differentiated hippocampal neurons. Treatment started at 7 DIC in the presence (A) or in the absence (B) of glia (see Materials and Methods). Survival was evaluated 4, 5, 6, and 12 d after the addition of gp120 in A and after 4, 6, and 9 d in B. Data shown in the figure are mean ± SEM and come from three different experiments for each treatment. (*p < 0.05 vs control).

Fig. 2.

Alterations in the nuclear density of neurons during treatment with gp120. Neurons are stained with fluorescein diacetate (yellow/green), propidium iodide (red), and Hoechst 33342 (white/blue). After 72 hr of treatment, the first changes in the nuclei, which are still negative to propidium iodide, are visible (A–D, large arrows). At this stage, blebs on the neurites are also present (C, small arrow). Dead neurons (not stained with fluorescein diacetate; A, B, small arrows) are stained with both propidium iodide and Hoechst 33342 and display either an enlarged (E, F, smallarrows) or a fragmented (E, F,large arrows) nucleus. Note that nuclei stained with both dyes are brighter (white) than normal nuclei, which are stained only with Hoechst 33342 (blue) (magnification, 500×).

Fig. 3.

In situ DNA fragmentation induced by gp120 in hippocampal neurons assessed by the TUNEL technique.A, Control neurons at 11 DIC; B, gp120-treated neurons (20 pm); C, gp120/TGF-treated (5 ng/ml) neurons. Treatments were started at 7 DIC.

Fig. 4.

Effect of the NMDA receptor antagonist APV (25 μm) and the protein synthesis inhibitor CHX (0.5 μg/ml) on gp120-induced neurotoxicity of mature hippocampal cultures. Treatments started at 7 DIC, and survival was assessed after 5 d. Data are expressed as mean ± SEM from three experiments (*p < 0.05 vs control).

Fig. 5.

Typical NMDA-evoked (50 μm) [Ca²⁺]_i increases in control neurons (A) and in gp120-treated neurons (B). Experiments were performed at 10 DIC in the presence of glycine (10 μm) and Mg²⁺(see Materials and Methods). Treatment with gp120 (20 pm) started at 7 DIC. Representative traces from control and treated neurons are shown in the figure.

Fig. 6.

Representative traces of the effect of two consecutive NMDA stimuli on [Ca²⁺]_i in control (A, B) and gp120-treated (C,D) neurons. The second NMDA application (B,D) occurs 15 min after washout of the first challenge.

Fig. 7.

Effect of TGF-β1 (5 ng/ml) on gp120-induced neurotoxicity. Data are from three different experiments and show the percentage of survived cells after 5 d of treatment (mean ± SEM; *p < 0.05 vs control). Treatments with vehicle, gp120, and TGF-β1 started at 7 DIC.

Fig. 8.

Inhibition of gp120-induced apoptosis (20 pm for 5 d) by TGF-β1 (5 ng/ml). The number of apoptotic cells was assessed by in situ detection of fragmented nuclei. Data are mean ± SEM (*p < 0.05 vs gp120 alone) from three different neuronal preparations.

Fig. 9.

Representative traces showing the increase in [Ca²⁺]_i induced by NMDA (50 μm) in neurons treated for 3 d with 20 pm gp120 in the absence (B) or in the presence (C) of TGF-β1 (5 ng/ml). A “peak and plateau” response similar to the one observed in control neurons (A) was found in the presence of TGF-β1.