Pregnenolone sulfate and cortisol induce secretion of acyl-CoA-binding protein and its conversion into endozepines from astrocytes

Abstract

Acyl-CoA-binding protein (ACBP) functions both intracellularly as part of fatty acid metabolism and extracellularly as diazepam binding inhibitor, the precursor of endozepine peptides. Two of these peptides, ODN and TTN, bind to the GABA(A) receptor and modulate its sensitivity to gamma-aminobutyric acid (GABA). We have found that depolarization of mouse primary astrocytes induces the rapid release and processing of ACBP to the active peptides. We previously showed that ODN can trigger the rapid sporulation of the social amoeba Dictyostelium. Using this bioassay, we now show that astrocytes release the endozepine peptides within 10 min of exposure to the steroids cortisol, pregnenolone, pregnenolone sulfate, or progesterone. ACBP lacks a signal sequence for secretion through the endoplasmic reticulum/Golgi pathway and its secretion is not affected by addition of brefeldin A, a well known inhibitor of the classical secretion pathway, suggesting that it follows an unconventional pathway for secretion. Moreover, induction of autophagy by addition of rapamycin also resulted in rapid release of ACBP indicating that this protein uses components of the autophagy pathway for secretion. Following secretion, ACBP is proteolytically cleaved to the active neuropeptides by protease activity on the surface of astrocytes. Neurosteroids, such as pregnenolone sulfate, were previously shown to modulate the excitatory/inhibitory balance in brain through increased release of glutamate and decreased release of GABA. These effects of steroids in neurons will be reinforced by the release of endozepines from astrocytes shown here, and suggest an orchestrated astrocyte-neuron cross-talk that can affect a broad spectrum of behavioral functions.

FIGURE 1.

Detection of mammalian endozepines using the Dictyostelium bioassay. A, synthetic TTN was added to the test cells at the indicated concentrations in the absence (filled circles) or presence (open circles) of anti-ACBP antibodies (1/5,000 final dilution). The proportion of spores was scored microscopically 1 h later. Each assay was repeated 5 times and the results averaged. The error bars correspond to 1 standard deviation. B, recombinant Hs ACBP was treated with trypsin overnight followed by purification on anion exchange resin. The indicated concentration of trypsinized ACBP was added to the test cells at the indicated concentration in the absence (filled circles) or presence (open circles) of anti-ACBP antibodies (1/5,000 final dilution). Unprocessed recombinant Hs ACBP was also added to the test cells (triangles). Each assay was repeated 5 times and the results averaged.

FIGURE 2.

Induction of endozepine production by KCl. Astrocytes in 24-well plates were washed twice with HSCC buffer and incubated in 500 μl of HSCC buffer at 37 °C. Endozepine production was induced by adding 50 mm KCl to the astrocytes (triangles). 2 μm TPCK (open squares) or anti-ACBP antibodies (diamonds) diluted 1/5,000 were added just prior to induction by 50 mm KCl. At the indicated times, 400 μl of buffer was harvested to quantify the amount of endozepines using the Dictyostelium bioassay. Each experiment was repeated at least three times. To standardize the experiments, cells from a well in each series were lysed using Triton X-100 and the amount of soluble protein measured. The bars indicate the reliability range.

FIGURE 3.

Induction of endozepine production by steroids. A, induction by pregnenolone sulfate and cortisol in the presence or absence of mifepristone. The indicated concentrations of cortisol (filled triangles) or pregnenolone sulfate (open circle) were added to astrocytes. For competition assay, 1 μm mifepristone was added to astrocytes just prior to endozepine induction with the indicated amount of cortisol (open circle) or pregnenolone sulfate (filled circle). The cells were incubated 15 min at 37 °C before harvesting 400 μl of the buffer to quantify the amount of endozepines with the Dictyostelium bioassay. The experiments were repeated at least three times. The bars indicate the reliability range. B, time course of endozepine production. Endozepine production was induced by addition of 100 nm cortisol to the astrocytes (circles) incubated at 37 °C for the indicated time. 2 μm TPCK (squares) or anti-ACBP antibodies (triangles) diluted 1/5,000 were added just prior to induction by cortisol. The amount of endozepine produced in a well was quantified. The experiments were repeated at least three times. The bars indicate the reliability range. C, induction by pregnenolone and progesterone. The indicated concentrations of pregnenolone (filled triangles) or progesterone (open square) were added to astrocytes. The amount of endozepine was quantified from samples harvested after 15 min incubation at 37 °C. The experiments were repeated at least three times. The bars indicate the reliability range.

FIGURE 4.

Processing of recombinant ACBP or ACBA by astrocytes. A, astrocytes were incubated with the indicated components: 20 pmol of ACBP for 1 h; 100 nm cortisol and 20 pmol of ACBP for 15 min; 100 nm cortisol for 15 min followed by two buffer changes (washed) and an additional 15-min incubation; 100 nm cortisol for 15 min followed by washing then incubation for 15 min in the presence of 20 pmol of ACBP with or without 2 μm TPCK. Endozepine activity was then quantified. The experiments were repeated at least three times, the bars indicate the reliability range. B, astrocytes were incubated with the indicated components: 20 pmol of Dictyostelium AcbA for 1 h; 100 nm cortisol for 15 min followed by two changes of buffer (washed) and an additional 15-min incubation in the presence or absence of 20 pmol of AcbA. The supernatant of astrocytes activated by cortisol was incubated for 15 min with 20 pmol of AcbA before determination of the level of SDF-2. The experiments were repeated at least three times.

FIGURE 5.

Induction of endozepine production by rapamycin. 2 μg/μl of rapamycin was added to astrocytes incubated at 37 °C in the absence (filled circles) or presence (open squares) of anti-ACBP antibodies. At the indicated time, 400 μl of buffer was harvested to quantify the amount of endozepines using the Dictyostelium bioassay. Each experiment was repeated at least three times. The bars indicate the reliability range.