The neurosteroids, allopregnanolone and progesterone, induce autophagy in cultured astrocytes

Abstract

Recent studies have suggested that neurosteroids such as pregnenolone, progesterone (PG) and their derivatives, have a role in activating autophagy in addition to diverse other functions. In our previous studies, we demonstrated that cellular free Zn²⁺ is involved in oxidative stress-induced autophagy and autophagic cell death in astrocytes. In the present study, we examined the possibility that neurosteroids, allopregnanolone (Allo) and PG, also activate autophagy in cultured mouse astrocytes through modulation of intracellular Zn²⁺.

Exposure of astrocytes to 250 nM Allo or 500 nM PG caused cytosolic vacuoles to appear within a few hours of treatment onset. Live-cell confocal microscopy of astrocytes transfected with red fluorescent protein-conjugated LC3 (RFP-LC3), a marker for autophagic vacuoles (AVs), as well as transmission electron microscopy, revealed that these vacuoles were AVs. In addition, Western blots showed increases in LC3-II levels. Interestingly, mTOR and Akt were concurrently activated, and their blockade further increased LC3-II levels and caused some cell death. These results indicate that co-activation of mTOR and Akt may act to limit neurosteroid-induced autophagy and thus inhibit autophagic cell death. As in other cases of autophagy, cellular Zn²⁺ levels increased after treatment with neurosteroids. The neurosteroid-induced increase in LC3-II levels was inhibited by addition of the Zn²⁺ chelator TPEN. Both the increase in LC3-II levels and activation of Akt and mTOR by neurosteroids were all mediated by PG receptors, as the effects were blocked by the addition of RU-486, a PG receptor antagonist. Moreover, mutant huntingtin (mHtt) aggregates in GFP-mHttQ74-transfected astrocytes were substantially reduced by neurosteroid treatment, indicating that neurosteroid-induced autophagy may be functional.

Present results demonstrate that Allo and PG activate autophagy in astrocytes. Notably, unlike several other autophagy inducers that, in excess, may cause autophagic cell death, Allo and PG are relatively non-toxic, possibly because of concurrent Akt and mTOR activation. Thus, as natural endogenous brain substances, Allo and PG may have a potential as therapeutic agents in neurodegenerative conditions in which abnormal protein aggregates are involved.

Introduction

Neurosteroids are steroid hormones that are synthesized in the central and peripheral nervous system, mainly by astrocytes. Cholesterol is the parent molecule of all neurosteroids, including pregnenolone, progesterone (PG), and their derivatives (Paul and Purdy, 1992). Although the precise functions of neurosteroids are still under investigation, some are already known. For instance, neurosteroids modulate inhibitory neurotransmission and regulate the growth of neurons (Grassi et al., 2007). Moreover, neurosteroids may have some neuroprotective effects (Wojtal et al., 2006). Whereas a GABA-potentiating effect has been speculated to underlie the neuroprotective effects of neurosteroids (Maitra and Reynolds, 1998), precise mechanisms have not yet been elucidated.

Recent studies have suggested that autophagy may function as a neuroprotective mechanism especially in chronic neurodegenerative conditions in which accumulation of abnormal proteins often plays a causal role. Macroautophagy or autophagy is an evolutionarily conserved cellular mechanism that degrades and recycles macromolecules and damaged organelles. Autophagosomes eventually fuse with lysosomes, and the inner membrane dissolves, resulting in the release of enveloped cytoplasmic contents into lysosomes and their degradation by lysosomal acidic hydrolases (Klionsky, 2007, Levine and Klionsky, 2004).

The PI3K/Akt/mTOR pathway is the best characterized regulatory pathway in autophagy. The protein kinase mTOR (mechanistic/mammalian target of rapamycin), a downstream target of PI3K/Akt, plays an essential role in regulating autophagy in response to nutrient deficiency, stress, and intracellular energy states. The phosphorylation and activation of mTOR via PI3K/Akt act to inhibit autophagy (Hay and Sonenberg, 2004). Akt is activated by diverse trophic receptors, including interferon growth factor receptor (IGFR), vascular endothelial growth factor receptor (VEGFR), and nerve growth factor receptor (NGFR) (Gerber et al., 1998, Nitta et al., 2004, Piiper et al., 2002). In addition to activating mTOR, Akt inhibits many forms of apoptosis (Maiuri et al., 2007).

Aberrant autophagy has been implicated in diverse human disorders, including cancer, inflammation, and neurodegeneration (Levine and Kroemer, 2008, Mizushima et al., 2008). In excess, autophagy may cause cell death via aggregation of abnormal protein (Anglade et al., 1997, Levine and Yuan, 2005). This mechanism has been proposed to contribute to neuronal death in acute brain injury (Smith et al., 2010). In contrast, inhibition of autophagy may cause the accumulation of diverse abnormal proteins and waste organelles, possibly resulting in chronic neurodegeneration or oncogenic transformation (Levine, 2007). Hence, in the latter context, boosting autophagy may help reduce pathological changes in certain disorders.

In our previous studies, we reported that changes in intracellular Zn²⁺ levels are closely tied with autophagy activation and lysosomal function. For instance, oxidative stress-induced autophagy and autophagic cell death in astrocytes are accompanied by a significant increase in the level of Zn²⁺ in the cytosol, and the formation of Zn²⁺-laden autophagic vacuoles (AVs) and lysosomes (Lee et al., 2009, Lee and Koh, 2010). Similar findings were observed in tamoxifen-induced autophagic death of MCF-7 breast cancer cells (Hwang et al., 2010) and clioquinol-induced autophagic death of astrocytes and neurons (Park et al., 2011). In these models, chelation of free Zn²⁺ resulted in almost complete blockade of autophagy and cell death. These findings led us to hypothesize that autophagy, in general, is regulated by intracellular free Zn²⁺ levels.

In the present study, we sought to determine whether (1) neurosteroids – allopregnanolone (Allo) and PG – activate autophagy in cultured astrocytes; and (2) if so, whether the resulting autophagy is modulated by changes in intracellular free-Zn²⁺ levels.