TSPO PIGA Ligands Promote Neurosteroidogenesis and Human Astrocyte Well-Being

Abstract

The steroidogenic 18 kDa translocator protein (TSPO) is an emerging, attractive therapeutic tool for several pathological conditions of the nervous system. Here, 13 high affinity TSPO ligands belonging to our previously described N,N-dialkyl-2-phenylindol-3-ylglyoxylamide (PIGA) class were evaluated for their potential ability to affect the cellular Oxidative Metabolism Activity/Proliferation index, which is used as a measure of astrocyte well-being. The most active PIGA ligands were also assessed for steroidogenic activity in terms of pregnenolone production, and the values were related to the metabolic index in rat and human models. The results showed a positive correlation between the increase in the Oxidative Metabolism Activity/Proliferation index and the pharmacologically induced stimulation of steroidogenesis. The specific involvement of steroid molecules in mediating the metabolic effects of the PIGA ligands was demonstrated using aminoglutethimide, a specific inhibitor of the first step of steroid biosynthesis. The most promising steroidogenic PIGA ligands were the 2-naphthyl derivatives that showed a long residence time to the target, in agreement with our previous data. In conclusion, TSPO ligand-induced neurosteroidogenesis was involved in astrocyte well-being.

Keywords: PIGA ligands; astrocytes; cellular proliferation; neurosteroidogenesis; oxidative metabolism; translocator protein.

Figure 1

Effects of the PIGA ligands on the Oxidative Metabolism Activity/Proliferation index in U87MG human glioma cells. U87MG cells were treated with different concentrations of the compounds (10 nM–1 µM) in serum-reduced media (1% fetal bovine serum (FBS)), and the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay was performed after 48 h of treatment. The data are expressed as percentages of the proliferative/oxidative metabolism activity index compared to the control (0.1% dimethyl sulfoxide (DMSO), a concentration that not interfered with the assay), which was set to 100%, and represent the means ± standard error of the mean (SEM) of three different experiments performed in duplicate. The statistical analysis was performed using one-way analysis of variance (ANOVA) and Bonferroni’s post-test; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control.

Figure 2

Effects of the PIGA ligands on pregnenolone production. C6 glioma cells (A) and U87MG cells (B) were incubated with different PIGA ligands (40 µM) for 2 h at 37 °C. The pregnenolone amounts were quantified by a competitive enzyme-linked immunosorbent assay. The data are expressed as percentage of pregnenolone production compared to the control, which was set to 100%, and represent the means ± SEM of three different determinations performed in duplicate. The statistical analysis was performed using one-way ANOVA and Bonferroni’s post-test; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control.

Figure 3

Spearman’s correlation analyses of the Oxidative Metabolism Activity/Proliferation (OMAP) index and pregnenolone production in U87MG cells. The percentages of pregnenolone production obtained in the steroidogenesis experiments (U87MG cells were exposed to 40 µM PIGA ligand in saline medium for 2 h) were correlated to the OMAP indexes obtained in the metabolic experiments (U87MG cells were exposed to 1 µM PIGA ligand in serum-reduced medium for 48 h). The statistical analyses were performed using the Spearman r correlation, reporting a p < 0.001.

Figure 4

Influence of pregnenolone production on the U87MG OMAP index. U87MG cells were treated with different concentrations of PIGA ligands (1 nM–10 µM) in the absence or presence of AMG (50 µM) in serum-reduced media (1% FBS), and the viable cells were counted after 48 h of treatment using the MTS assay. The data are expressed as percentages of the OMAP index compared to the control, which was set to 100%, and represent the means ± SEM of three different experiments performed in duplicate. The statistical analysis was performed using one-way ANOVA and Bonferroni’s post-test; ** p < 0.01, *** p < 0.001 vs. the control; ## p < 0.01, ### p < 0.001 vs. the respective treatment without dl-aminoglutethimide (AMG).

Figure 5

In vitro response of the human astrocytes to the PIGA1138 and PK11195 treatments. (A) Human astrocytes were treated with different concentrations of PIGA1138 and PK11195 (10 nM–1 µM) in serum-reduced media (1% FBS), and the MTS assay was performed after 48 h of treatment; (B) Human astrocytes were treated with different concentrations of PIGA1138 and PK11195 (10 nM–1·µM) in complete medium and the MTS assay was performed after 48 h of treatment. The data are expressed as percentages of metabolic activity compared to the control, which was set to 100%, and represent the means ± SEM of three different experiments performed in duplicate. ** p < 0.01, *** p < 0.001 vs. the control.