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View metadata, citation and similar papers at core.ac.uk brought to you by CORE REVIEW ARTICLE published: 28 December 2011 provided by PubMed Central doi: 10.3389/fendo.2011.00103 The allopregnanolone modulates specific functions in central and peripheral glial cells

Alessandro Faroni 1,2 and Valerio Magnaghi 2*

1 Blond McIndoe Laboratories, Regenerative Biomedicine, School of Medicine, The University of Manchester, Manchester, UK 2 Department of Endocrinology, Physiopathology, Applied Biology, University of Milan, Milan, Italy

Edited by: Since the first observations on the existence of “” in the 1980s, our under- Hubert Vaudry, University of Rouen, standing of the importance of these endogenous in the control of the central France and peripheral (PNS) has increased progressively. Although most of the Reviewed by: Ishwar Parhar, Monash University, observations were made in neuronal cells, equally important are the effects that neuros- Malaysia teroids exert on glial cells. Among the different classes of neurosteroids acting on glial Michael Schumacher, INSERM, cells, the 5α-3α , allopregnanolone, displays a particular mecha- France nism of action involving primarily the modulation of classic GABA receptors. In this review, *Correspondence: we focus our attention on allopregnanolone because its effects on the physiology of glial Valerio Magnaghi, Department of Endocrinology, Physiopathology, cells of the central and PNS are intriguing and could potentially lead to the development of Applied Biology, University of Milan, new strategies for and/or regeneration of injured nervous tissues. Via G. Balzaretti 9, 20133 Milan, Italy. Keywords: neuron–glial interaction, GABA, non-genomic action, myelin, Schwann cell e-mail: [email protected]

INTRODUCTION support neuron and survival, they can uptake and The first observations on the existence of “nervous steroids” produce and they are also able to synthesize appeared in the early 1980s, introducing the novel concept that neurosteroids (Celotti et al., 1992; Melcangi et al., 2001b). Glial some hormonal steroids may be synthesized de novo in the ner- cells, indeed, possess all the enzymatic pathways and the synthetic vous system. Baulieu (1997) coined the term “neurosteroids” in machinery required to produce steroids (that is the P450 choles- order to define such molecules. Over the years, the importance terol side-chain cleavage enzyme, P450SCC; 17α-hydroxysteroid- of such endogenous steroids in the control of both the central dehydrogenase, 17α-HSD; 3β-hydroxysteroid-dehydrogenase, 3β- nervous system (CNS) and peripheral nervous system (PNS) has HSD and other enzymes), or to convert them into neuroac- become increasingly apparent. However, most of the observations tive (Mellon et al., 2001). In particular, the enzy- were made through investigations into their role in neuronal cells. matic complex formed by the 5α-reductase (5α-R) and the 3α- Equally important are the putative effects that neurosteroids may hydroxysteroid-dehydrogenase (3α-HSD) enzymes has been char- directly exert on glial cells by regulating their physiological func- acterized in several areas of the human brain (Steckelbroeck et al., tions, or even indirectly through the complex interactions with 2001) and in the PNS (Melcangi et al., 1990b, 1992, 1999b; Celotti neuronal cells. et al., 1992). The combined action of these two enzymes catalyzes It is now generally believed that central (i.e., astrocytes, oligo- the conversion of some native steroids, such as progesterone (P), dendrocytes) and peripheral glial cells (i.e., Schwann cells, SC) into their more active 5α-3α-reduced metabolites, that is into are fundamental for the regulation of neuronal activity. They “neurosteroids.” This enzymatic complex is extremely versatile since every possessing the δ4-3keto configuration could potentially undergo the 5α-reduction and subsequently be sub- α Abbreviations: 3α-HSD, 3α-hydroxysteroid-dehydrogenase; 3βHSD, 3β- jected to 3 -hydroxylation (Celotti et al., 1992; Melcangi et al., hydroxysteroid-dehydrogenase; 5HT3, 5-hydroxytryptamine type 3; 17αHSD, 1999b, 2000c). By analyzing the ability of purified primary cell 17α-hydroxysteroid-dehydrogenase; ALLO, allopregnanolone; AR, cultures of neurons, astrocytes (type 1 and type 2 astrocytes) or ; bFGF, basic fibroblast growth factor; CMT1A, Charcot Marie Tooth type oligodendrocytes to metabolize (T; Melcangi et al., 1A; CNPase, 2-3-cyclic nucleotide-3-phospodiesterase; CREB, cAMP response element-binding protein; DHP, dehydroprogesterone; EAAC1, excitatory amino 1990a, 1993, 1994), it has been demonstrated that neurons pos- acid transporter 1; ER, receptor; GABA, γ-aminobutyric acid; GABA-A, sess significantly higher amounts of 5α-R activity compared to GABA type A receptor; GABA-B, GABA type B receptor; GAD67, astrocytes and oligodendrocytes (Melcangi et al., 1993, 1994). decarboxylase of 67 kDa; GFAP, glial fibrillary acid protein; IGF-I, insulin-like Interestingly, the simultaneous presence of neurons and type 1 growth factor-I; LHRH, luteinizing-hormone-releasing hormone; MAG, myelin astrocyte cultures stimulates the 5α-R activity (Melcangi et al., associated glycoprotein; MBP, myelin basic protein; mER, membrane estrogen receptor; mPR, membrane ; NMDA, N -Methyl-d-aspartate; 2001c),indicating a possible interaction of these cells in the metab- P450SCC, P450 side-chain cleavage enzyme; P,progesterone; P0, myelin olism of neurosteroids. It is also reasonable to speculate that the protein zero; PK-A, protein kinase A; PK-C, protein kinase C; PMP22, peripheral metabolism of neurosteroids may be relevant for differentiation myelin protein of 22 kDa; PNS, peripheral nervous system; PR, progesterone α + of glia. Gago et al. (2001) showed that the formation of the 5 - receptor; PSA-NCAM , polysialylated-neural cell adhesion molecule positive cells; reduced metabolite of P, dehydroprogesterone (DHP), is fivefold SC, Schwann cells; 5α-R, 5α-reductase; T, testosterone; TGFα, transforming growth factor α;TGFβ1, transforming growth factor β1; TGFβ2, transforming growth higher in fully differentiated oligodendrocytes compared to oligo- factor β2; TNFα, tumor necrosis factor α. dendrocyte progenitor cells. This suggests that the acquisition

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of the neurosteroids biosynthetic capacity is a marker of glial is represented by the ALLO’s activation of GABA-A receptor differentiation. In addition, the reaction catalyzed by 3α-HSD functions (Majewska et al., 1986; Rupprecht and Holsboer, 1999; involves the replacement of a carbonyl with a hydroxyl group,lead- Lambert et al., 2003). Interestingly, ALLO’s mechanism of action ing to the formation of metabolites that are potent modulators on GABA-A receptor is dependent. Indeed, in of non-classical steroid receptors (see below). Some biochemi- the low nanomolar aqueous concentration ALLO acts alloster- cal studies demonstrated that 3α-HSD is distributed in various ically, enhancing the action of the natural GABA, while regions of the rodent brain (Khanna et al., 1995; Mellon et al., at higher concentration (micromolar range) ALLO directly gates 2001; Agis-Balboa et al., 2007), where it is mainly localized in type the GABA-A receptor channel complex (Callachan et al., 1987; 1 astrocytes (Melcangi et al., 1993, 1994) and in oligodendrocytes Puia et al., 1990). However, it has been shown that neurosteroids (Melcangi et al.,1994). The 5α-R-3α-HSD activity is also present in may directly gate GABA-A receptors also at lower concentra- the SC of the PNS (Melcangi et al., 1990b, 1998, 1999b; Yokoi et al., tion, around 100 nM, but the kinetic of this receptor activation 1998). Therefore, the ability to synthesize or metabolize particular is relatively slow (Shu et al., 2004). arrays of steroids seems to be region-specific in the different CNS The GABA-A receptor is a member of the ligand-gated ion areas and confined to glial cells (Mellon et al., 2001). In line with channel family, permeable to chloride ions and composed of this hypothesis, the 5α-R activity in the myelin forming cells of five subunits drawn from a repertoire of 19 isoforms (that is the CNS and PNS (that is oligodendrocytes and SC, respectively) α1–6, β1–3, γ1–3, δ, ε, π, θ, ρ1–3; Whiting et al., 1995, 1997; appeared different, being about four times higher in SC compared Lambert et al., 2003). The GABA-A receptor is blocked by bicu- to oligodendrocytes (Melcangi et al., 1998). Thus, the presence of culline and , but channel opening is enhanced by the 5α-R-3α-HSD enzymatic complex in oligodendrocytes and SC , , and also neurosteroids suggests that the locally formed neurosteroids might play a crucial (Park-Chung et al., 1999; Belelli and Lambert, 2005). Interest- role in the process of myelination (Melcangi et al., 1988, 2001c; ingly, the GABA-A receptor modulation exerted by neurosteroids Martini et al., 2003). is enantioselective and is partially dependent upon the receptor The neurosteroid 5α-pregnan-3α-ol-20-one, also named subunit composition. For this reason several studies were aimed at tetrahydroprogesterone or allopregnanolone (ALLO) is the most elucidating the putative neurosteroids external binding sites (Lam- important hormonal steroid that was originally shown to act as bert et al., 2003). Although no special requirement concerning the a neurosteroid. It is synthesized through the action of the 5αR- nature of the α and β subunits seems to be essential to confer sen- 3α-HSD, which converts P into DHP and subsequently, via a sitivity to neurosteroids (Belelli et al., 2002), a receptor composed bidirectional reaction, into ALLO (see Figure 1, in SC). Interest- of one α (α2–5) assembled with β3 and γ2S subunits gives con- ingly,ALLO is able to modulate several neuronal cell functions. For sistent potentiation of ALLO-mediated GABA-activated currents instance, it is proven to be a neurogenic molecule, inducing a dose (Hosie et al., 2009). Moreover, γ2 subunits confer more sensitiv- dependent significant increase in proliferation of rat neural prog- ity than γ1orγ3(Belelli et al., 2002), while δ subunit potentiates enitor cells and of human neural stem cells (Wang et al., 2005), or the action of the 5α-3α-reduced neurosteroids (Mihalek et al., affecting cerebellar neurogenesis (Keller et al., 2004). Besides these 1999; Belelli et al., 2002). The extrasynaptic GABA-A receptor studies revealing ALLO’s actions on the neuronal compartment, incorporating the δ subunit, indeed, is an important and highly several observations have suggested important roles for ALLO on sensitive target of neurosteroids (Lambert et al., 2009), implicating glial cells. In this review we attempt to summarize the intrigu- the δ-containing receptors as the preferred target for the action of ing modulations and the therapeutic potential of the neurosteroid neurosteroids. Nevertheless,the δ subunit seems to primarily influ- ALLO on the glial cells of the CNS and PNS. ence the transduction of neurosteroid signals rather than engage directly in binding (Hosie et al., 2009). MECHANISM OF ACTION OF NEUROSTEROIDS Neurosteroids might also indirectly target the receptor function Neurosteroids exert complex effects in the nervous system through by regulating the transcription of some GABA-A receptor sub- “classic” and “non-classic” actions. The “classic” genomic action units, thus altering the GABA-A inhibitory activity for a prolonged consists of the modulation of their target cells by regulating gene time (Maguire and Mody,2009). Neurosteroids might also interact transcription after binding to nuclear receptors (Slater et al.,1994). with protein kinase or phosphatases, which in turn act on GABA-A By contrast, the rapid “non-classic” action involves the modula- subunits altering the entire receptor function (Belelli and Lambert, tion of receptors, that is γ-aminobutyric acid 2005). Likewise, phosphorylation of GABA-A receptors by pro- (GABA), N -Methyl-d-aspartate (NMDA), 5-hydroxytryptamine tein kinase C (PK-C) influences the sensitivity to neurosteroids type 3 (5HT3) and σ-receptors (Lambert et al., 1996; Rupprecht (Brussaard and Koksma, 2003; Vergnano et al., 2007). et al., 2001; Monnet and Maurice, 2006; Sedlacek et al., 2008) Beside the external binding sites of GABA-A receptor, and the activation of novel putative membrane receptors for some recent evidence supports a novel hypothesis suggesting steroids (Hammes and Levin, 2007; Dressing et al., 2011; Levin, that neurosteroids require a membranous route of access to 2011). Some of these receptors have been found in glial cells (see transmembrane-domain binding sites within GABA-A receptor Tables 1–3). (Shu et al., 2004; Akk et al., 2005; Hosie et al., 2006; Chisari et al., Allopregnanolone is a potent modulator of the GABA type 2009). Once neurosteroids reach the site of action they may dif- A (GABA-A) receptor (Majewska et al., 1986; Majewska, 1992; fuse through the plasma membrane to directly bind the GABA-A Lambert et al., 1995). In fact, the most important and well- receptor, rather than interacting with external binding sites of characterized “non-classic,” non-genomic action of neurosteroids the receptor. This is a low-affinity receptor interaction that might

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depend on the lipid composition of the cell membrane. Neuros- ALLO’s EFFECT ON ASTROCYTE teroids may be also retained in the intracellular compartment and Neurosteroids are known to control the proliferation, cell shape, then re-supply the plasma membrane with ligands able to mod- and gene expression of CNS astroglia (Garcia-Segura et al., 1996, ulate the GABA-A receptor at later times, so that the rate of this 1999; Jordan, 1999; Nichols, 1999). The cytoskeletal protein, glial transport can alter the kinetics of neurosteroids actions (Akk et al., fibrillary acidic protein (GFAP) is usually responsible for modulat- 2005). ing astrocyte shape and motility (Laping et al., 1994). Many of the The proposed mechanisms of ALLO’s action, however, may effects of neurosteroids on the expression and synthesis of GFAP be in principle complementary to the classic genomic effects. are due to their active metabolites. For instance, the gene expres- Indeed, an indirect action of ALLO involving the classic prog- sion of GFAP in culture of astrocytes is inhibited by ALLO and esterone receptor (PR) should not be totally excluded. In such a stimulated by DHP, suggesting that different intracellular signal- scenario, ALLO’s action closely depends on its retro-conversion to ing pathways are likely to be involved in astrocytes proliferation DHP, operated by enzyme 3α-HSD (Martini et al., 2003), which (Melcangi et al., 1996). Nevertheless, it has been proposed that may then stimulate the PR generating a classic genomic effect. In the ALLO’s effects on astrocytes are mediated by the activation of comparison to ALLO’s rapid interaction with GABA-A receptors, GABA-A receptors, whose subunits are widely distributed in astro- the PR-mediated mechanism shows slower kinetics and lasts for a cytes (Bovolin et al., 1992; Hosli et al., 1997; Israel et al., 2003). longer time. The effects of neuroactive steroids have also been evaluated on Finally, concerning the ALLO interaction with the GABAer- ammonia-induced astrocyte swelling in culture, a phenomena that gic system, its putative ability to interact with the metabotropic occurs early after some acute CNS pathologies such as ischemia GABA-B receptor should not be totally excluded, although and traumatic brain injury. Bender and Norenberg (1998) showed the evidence that such an interaction effectively occurs is not that nanomolar concentration of ALLO diminishes the astrocyte clearly provided. GABA-B receptor is a distinct, -sensitive, swelling, likely via a GABA-A mechanism. These observations metabotropic receptor that is a member of the seven transmem- might be very useful for the development of new therapeutic brane G-protein-coupled receptor superfamily. GABA-B receptors approaches to treat the acute hyperammonemic syndromes and may influence the activity of adenylate cyclase and/or modu- other associated pathological conditions (Bender and Norenberg, late calcium and potassium channels activity (Bowery and Enna, 1998). 2000). In this regard, we have recently demonstrated in vitro Allopregnanolone seems to be also involved in the feedback a cross-correlation between the GABA-A and B receptors in mechanisms regulating the gonadal axis, and in particular the SC (Magnaghi, 2007), and some of these effects are discussed neuroendocrine function of the luteinizing-hormone-releasing below. hormone (LHRH). Herbison and collaborators provide the first

Table 1 | Distribution of major classic and non-classic receptors for neurosteroids in astrocytes.

Classic intracellular receptors Non-classic membrane receptors

PR ER AR GABA-A NMDA mER mPR

Rat glial Pig brain areas Primate Mouse corpus callosum Rat celebral cortex (Conti et al., Rat glial Rat spinal culture (Langub and prefrontal cortex (Berger et al., 1992) 1996), spinal cord slice (Ziak culture (Kuo cord (Labom- (Jung-Testas Watson, 1992) (Finley and et al., 1998), and injured rat et al., 2011) barda et al., et al., 1992) Kritzer, 1999) hippocampus (Krebs et al., 2003) 2011) Rat brain Rat glial culture Rat hypothalamus (Israel Human celebral cortex (Conti (Azcoitia et al., (Jung-Testas et al., 2003), hippocampus et al., 1999) 1999; Cardona- et al., 1992) (Macvicar et al., 1989; Fraser Gomez et al., et al., 1995; Kang et al., 1998) 2000; Milner spinal cord (Chvatal et al., et al., 2001) 1995; Pastor et al., 1995), optic nerve astrocytes (Butt and Jennings, 1994) Rat glial Rat glial culture (Kettenmann Mouse cortex (Lalo et al., 2006) culture et al., 1987; Backus et al., and neocortex (Schipke et al., (Jung-Testas 1988; Bormann, 1988; 2001) et al., 1992; Rosewater and Sontheimer, Santagati 1994; Hosli et al., 1997; et al., 1994) Matsutani and Yamamoto, 1997) Human brain and human glial Human glial culture (Lee et al., culture (Lee et al., 2011a,b) 2011a,b)

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Table 2 | Distribution of major classic and non-classic receptors for neurosteroids in oligodendrocytes.

Classic intracellular receptors Non-classic membrane receptors

PR ER AR GABA-A NMDA mER mPR

Rat glial Rat glial Primate Mouse corpus callosum (Berger Rat spinal cord slice (Ziak et al., – Rat spinal culture culture prefrontal et al., 1992) 1998), cerebellum and corpus cord (Jung- (Jung-Testas cortex (Finley callosum slices (Karadottir et al., (Labom- Testas et al., 1992; and Kritzer, 2005), and optic nerve (Salter and barda et al., et al., 1992) Santagati 1999) Fern, 2005; Micu et al., 2006) 2011) et al., 1994) Rat glial Rat spinal cord (Pastor et al., 1995) Rat glial culture (Wang et al., 1996) culture and optic nerve (Borges et al., 1995) (Jung-Testas Mouse glial culture (Gilbert et al., et al., 1992) 1984a; Kettenmann et al., 1984; Hoppe and Kettenmann, 1989) Culture of mouse (Von Blankenfeld et al., 1991; Kirchhoff and Kettenmann, 1992) and rat (Belachew et al., 1998; Williamson et al., 1998) precursor cells.

Table 3 | Distribution of major classic and non-classic receptors for neurosteroids in Schwann cells.

Classic intracellular receptors Non-classic membrane receptors

PR ER AR GABA-A NMDA mER mPR

Rat SC in culture (Jung-Testas et al., Rat SC in culture Rat SC in culture Rat SC in culture Guinea pig SC in –– 1996a; Jung-Testas and Baulieu, 1998; (Jung-Testas and (Magnaghi et al., 1999) (Melcangi et al., 1999a; culture (Fink Thi et al., 1998; Magnaghi et al., 1999) Baulieu, 1998) Magnaghi et al., 2006) et al., 1999) Rat sciatic nerve (Magnaghi et al., Rat sciatic nerve Giant squid SC 2001) (Melcangi et al., 1999a; (Evans et al., Magnaghi et al., 2006) 1991, 1992) evidence for a direct action of ALLO on postnatal LHRH neu- ALLO’s action may imply an indirect mechanism mediated via PR rons, suggesting a new mechanism by which fluctuating P levels (Melcangi et al., 2001a). may influence the secretory activity of these neurons in the female hypothalamus (Sim et al., 2001). At the cellular level, the increased ALLO’s EFFECT ON OLIGODENDROCYTE ALLO concentration, typical of late , has important In mature oligodendrocytes (the cells responsible for forming the physiological actions in repressing the electrical activity of specific central myelin) the progestagens neurosteroids regulate the gene magnocellular oxytocin neurons in the hypothalamus. Moreover, expression of some important proteins, such as the myelin basic the fall in ALLO concentration prior to parturition equally impacts protein (MBP) and the 2 -3 -cyclic nucleotide-3-phospodiesterase GABA-A receptor signaling in oxytocin neurons, as well as in the (CNPase; Verdi and Campagnoni, 1990; Jung-Testas et al., 1996b). hippocampus and frontal cortex neurons, suggesting that ALLO’s The age-associated decline in MBP expression levels in 22- to 24- concentration changes during pregnancy are likely to exert a pow- month-old male rats, for instance, was reversed by ALLO and DHP, erful regulatory influence upon neurotransmission in a variety of still indicating a PR-mediated genomic mechanism (Ibanez et al., brain networks (Herbison, 2001). 2003). The progestagens involvement on MBP expression regu- However, paracrine mechanisms involving astrocyte cells may lation in oligodentrocytes was demonstrated also in organotypic also be at the base of positive or negative feedback signals regu- slice cultures of 7-day-old rat and mouse cerebellum (Ghoumari lating the reproductive axis. Indeed, growth factors such as trans- et al., 2003). This effect likely involves classic PR, since it was forming growth factor α (TGFα), transforming growth factor β1 mimicked with the selective PR R5020 and blocked with (TGFβ1) and β2(TGFβ2), as well as basic fibroblast growth factor the PR antagonist (Ghoumari et al., 2003). However, (bFGF) and insulin-like growth factor-I (IGF-I) are released by ALLO significantly stimulates MBP expression in 7-day-old rat astrocytes and participate in the control of LHRH release (Mel- and mouse cerebellum cultures, and this effect seemed to be medi- cangi et al., 1997; Ojeda and Ma, 1999). ALLO stimulates TGFβ1 ated via GABA-A receptor activation. In accordance, the GABA-A gene expression in hypothalamic astrocytes, nevertheless also P antagonist counteracts the ALLO stimulatory effect, and DHP proved able to exert an identical effect, suggesting that confirming a GABA-A-mediated mechanism (Ghoumari et al.,

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2003). It is important to underline that some GABA-A receptor parameters of the PNS (Melcangi et al., 2000c; Magnaghi et al., subunits are expressed in oligodendrocytes (Gilbert et al., 1984b) 2001). For instance, progestagens stimulated the expression of spe- and in oligodendrocyte progenitor cells (Kettenmann et al., 1984; cific peripheral myelin proteins, such as the myelin protein zero Kirchhoff and Kettenmann, 1992). Gago et al. (2004) observed (P0) and the peripheral myelin protein 22 (PMP22), in the sciatic that the neuroactive steroids and GABA signaling are responsible nerve of young and old male rats (Melcangi et al., 1998, 1999a, for the autocrine/paracrine loops controlling neuronal progen- 2000c; Magnaghi et al., 2001). Moreover, they reduced myelin itors proliferation and differentiation. The neuronal progenitor abnormalities and fibers loss in aged sciatic nerve (Azcoitia et al., polysialylated-neural cell adhesion molecule positive cells (PSA- 2003), promoting re-myelination after cryolesion (Koenig et al., NCAM+), indeed, possess several GABA-A receptor subunits 1995), transection (Melcangi et al., 2000a), or crush injury (Roglio (Nguyen et al., 2003; Gago et al., 2004), like the α1-5, β2, β3, and et al., 2008). Mifepristone (RU38486), a classic PR antagonist, was γ2 isoforms (Gago et al., 2004). Interestingly, GABA increased in recently studied because of its ability in decreasing the PMP22 a dose dependent manner the proliferation of these cells and the overexpression in a rat model of peripheral inherited neuropa- effect was bicuculline sensitive, suggesting a GABA-A-mediated thy (i.e., the Charcot Marie Tooth type 1A, CMT1A; Sereda et al., mechanism. The effect on the PSA-NCAM+ proliferation was 2003). These results lead to the conclusion that PR likely partic- also mimicked by nanomolar concentration of ALLO. Moreover, ipates in the control of specific peripheral myelin proteins, such P via its conversion in ALLO, proved able to stimulate the early as P0 and PMP22 (Magnaghi et al., 2001). Nevertheless, in vitro PSA-NCAM+ progenitor proliferation (Gago et al., 2004). studies showed that ALLO, besides the ability of increasing P0 Collectively, these findings suggest a pivotal role of ALLO and mRNA levels, is the main neurosteroid able to stimulate the lev- GABA in the development and maturation of oligodendrocytes els of PMP22 mRNA and protein (Melcangi et al., 1999a, 2000b; (Ben-Ari, 2002; Gago et al., 2004). Magnaghi et al., 2001). Allopregnanolone is a potent modulator of GABA-A receptor, ALLO’s EFFECT ON MICROGLIA which subunits are widely distributed in SC (Melcangi et al.,1999a; Steroids exert anti-inflammatory actions in the peripheral tissues Magnaghi et al., 2006). SC in culture, indeed, express the messen- as well as in the brain, where they act mainly on microglial cells. gers for several GABA-A receptor subunits, that is α 2 and 3, β 1, In this regard, some efforts were addressed to study the neuro- 2, and 3 (Melcangi et al., 1999a; Magnaghi et al., 2006). protective role of and progesterone on microglial cells Therefore,to investigate if the effect of ALLO on PMP22 expres- (Stone et al., 1997; Vegeto et al., 1999, 2001; Drew and Chavis, sion was mediated by interaction with GABA-A receptors, ALLO’s 2000; Bruce-Keller et al., 2001). In parallel, several line of evi- stimulation was mimicked with specific GABA-A ligands. Bicu- dence showed that ALLO plays a neuroprotective role following culline, a specific GABA-A antagonist, abolishes the effect of ALLO different brain injury conditions, such as ischemic damage (Kelley on PMP22 expression levels, whereas (a specific GABA- et al., 2011), PTX-induced seizures (Singh et al., 2010) and oxygen- A agonist) mimics ALLO’s action (Magnaghi et al., 2001, 2006), glucose deprivation (Ardeshiri et al., 2006). The of confirming the hypothesis that the expression of PMP22 in SC is ALLO are also remarkably high in the fetal brain and further rise under the control of GABA-A receptors. However, ALLO’s action in response to acute hypoxia, representing an endogenous protec- via the GABAergic system in SC is even more complex, because tive mechanism in the developing brain (Hirst et al., 2006). These it also involves the GABA-B receptor and the endogenous GABA effects might be generally ascribed to ALLO’s regulation of anti- neurotransmitter (see Figure 1 for details). inflammatory cytokines and endogenous antioxidants, although Recent observations obtained by reverse transcriptase- only few evidences rely on the direct effect of ALLO on microglial polymerase chain reaction, western blot and immunohistochem- cells. For instance, the endogenous concentration of ALLO in istry analyses confirmed the presence of GABA-B receptor sub- the brain is increased concomitantly to the lipopolysaccharides- units (1a, 1b, and 2) in SC, where the functional receptor was induced tumor necrosis factor α (TNFα) production, which in proved to be negatively coupled to the adenylate cyclase system turn inhibits TNFα levels (Ghezzi et al., 2000). ALLO also protects (Magnaghi et al., 2004). The activation of the GABA-B receptor against apoptotic cell death in an in vivo model of rat prefrontal in SC influences cell proliferation, as demonstrated with baclofen cortex injury, even if this effects were not totally referred to a treatment that counteracted the forskolin-induced SC prolifer- microglial cell disregulation (Djebaili et al., 2005). ation at 5 days in vitro (Magnaghi et al., 2004); at the same time point also the percentage of BrdUrd immunopositive SC ALLO’s EFFECT ON SC was reduced (Magnaghi et al., 2004). Since the activation of Over the last few decades a large body of evidence has accumu- the adenylate cyclase system potentially regulates SC prolifera- lated from investigations into the potential effects that neuros- tion (Lee et al., 1999; Mirsky and Jessen, 1999), we speculated teroids exert on SC, which are the cells ensheathing axons and that GABA-B-induced decrease in cAMP levels may affect the forming the myelin in the PNS (Jessen and Mirsky, 1997). The cell proliferation (Magnaghi et al., 2004). Notably, changes in first studies started when Baulieu and colleagues focused their cAMP levels in SC may also influence the expression of specific attention on neurosteroid , analyzing primarily the myelin proteins (Suter et al., 1994; Scherer et al., 1995; Lee et al., peripheral nerves, such as the sciatic nerve, and the SC (Jung- 1999). In agreement, GABA-B activation decreases the expression Testas et al., 1993). The progestagens, P and its 5α-derivatives of some important myelin proteins, like P0, PMP22 as well as other (DHP and ALLO) have been studied in vivo, demonstrating that proteins, like connexin 32 and the myelin associated glycoprotein they are able to modulate several biochemical and morphological (MAG; Magnaghi et al., 2004).

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Investigating the putative effects of neurosteroids on GABA- Most of the effects exerted by ALLO in SC were achieved at B receptor subunits expression in the SC in vitro, it was also nanomolar concentration, involving an allosteric interaction that shown that ALLO controls the expression of different GABA-B necessarily entails the presence of the endogenous ligand GABA. receptor subunits (Magnaghi et al., 2006). The expression pro- To make the issue even more puzzling, the presence of GABA and file was biphasic; after 4 h of exposure ALLO produces a robust its synthetic machinery was thus demonstrated in SC. In particular stimulation of the mRNA of all three subunits, -1a, -1b, and - the glutamic acid decarboxylase of 67 kDa (GAD67), a key enzyme 2, while at 24 h of exposure ALLO decreased the expression of for GABA synthesis, was shown to be expressed in SC. Acting in a -1b and -2 subunits (Magnaghi et al., 2006). Since ALLO’s effect way that resemble an autocrine loop, ALLO (10 nM) increased the on GABA-B receptor expression was mimicked by muscimol and levels of GAD67 in SC, thus stimulating GABA synthesis and pro- GABA, it was hypothesized that ALLO regulates GABA-B recep- viding the natural ligand for GABA-A receptor (Magnaghi et al., tor via a GABA-A-mediated mechanism (Magnaghi et al., 2006; 2010). Magnaghi, 2007). The intracellular pathways downstream of this Finally, in line with this hypothesis, it was recently demon- interaction is rather complicated and presently not completely strated that SC in vitro possess a functionally active glutamate identified, however a possible hypothesis is proposed below. After uptake system able to provide glutamate as a precursor for the 24 h of exposure, P and DHP give rise to a GABA-B receptor mod- synthesis of GABA (Perego et al., 2011). Indeed, it was first demon- ulation similar to that induced by ALLO. The longer time required strated that SC express the excitatory amino acid transporter 1 leads to the hypothesis that P and DHP effects are due to their (EAAC1) in the plasma membrane and in intracellular vesicular 5α-R-3α-HSD mediated conversion into ALLO (Magnaghi et al., compartments of the endocytic recycling pathway. Interestingly, 2006; Magnaghi, 2007). Therefore, GABA-B subunits expression EAAC1 activity can be modulated by exposure of SC to ALLO may be differently influenced, mainly by ALLO via a GABA-A- 10 nM. The transporter up-regulation by ALLO was rapid, did mediated mechanism, but also by its precursors P and/or DHP not involve protein neo-synthesis and was prevented by actin after 5α-R-3α-HSD conversion into ALLO. depolymerization. The modulation exerted by ALLO involved

FIGURE1|Schematic representation showing ALLO, GABA and its on the SC surface (Magnaghi et al., 2006). This modulates the expression receptors (GABA-A and GABA-B) acting in the bidirectional cross-talk and the responsiveness of the GABA-B receptor (Magnaghi et al., 2006, between neurons and SC. We propose the following hypothesis on the 2010), and in turn its desensitization. Contemporarily, ALLO stimulates the functional role of the GABAergic system in SC. GABA, coming from the SC proliferation (Perego et al., 2011), inducing other genomic effects such neuronal compartment or produced by the SC (Magnaghi et al., 2010), as the increase of myelin proteins (Melcangi et al., 1999a; Magnaghi may affect the paracrine cross-talk between these cells. The extracellular et al., 2001), and the rising of GAD and GABA levels (Magnaghi et al., GABA might interact with the GABA-B receptor on the SC surface, 2010). The last effect, being a sort of autocrine mechanism, involving decreasing their proliferation (Magnaghi et al., 2004). This challenge then enhanced cAMP levels and the PK-A pathway. Collectively, these findings diminishes the cAMP levels, reducing the myelin proteins expression suggest that an autocrine mechanism involving ALLO and GABA is (Magnaghi et al., 2004) and prompts the SC to start differentiation. The particularly relevant for the control of SC proliferation/differentiation, and neurosteroid ALLO, which is produced by SC (Baulieu and Schumacher, may be considered the result of a balanced activation of GABA-A or 1997; Melcangi et al., 1999b) allosterically activates the GABA-A receptor GABA-B receptors on SC.

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GABA-A receptor and PK-C activation, promoting the exocyto- proteins expression in SC. Muscimol mimicked this effect on SC, sis of the EAAC1 transporter from intracellular stores to the SC further supporting that GABA-A might also participate in the membrane (in actin-rich cell tips), and modifying the morphol- control of the SC proliferation/differentiation (Figure 1). ogy of SC processes. The evidence that EAAC1 transporter controls However, the nature of the signaling pathways downstream the ALLO-mediated effects on SC proliferation was also provided to the ALLO’s allosteric modulation of GABA-A receptor is still (Perego et al., 2011). debated. We suggest an involvement of the protein kinase A (PK- A) pathway, which through enhanced cAMP levels and CREB DOWNSTREAM MECHANISM FOLLOWING ALLO’s phosphorylation, modulates the allosteric action of ALLO at the ACTIVATION OF GABA-A RECEPTOR: IS THE PUZZLE GABA-A receptor (Magnaghi et al., 2010). However, other protein UNPUZZLED? kinases should be also considered. In fact, the allosteric ALLO’s Several attempts in the literature tried to explain the intracellular modulation of GABA-A receptor, regulating glutamate uptake via molecular mechanism downstream to ALLO’s allosteric activa- EAAC1 transporter, is dependent by a PK-C activation, as con- tion of GABA-A receptor, in neurons as well as in central and/or firmed by the very recent analysis of the pharmacological profile peripheral glial cells. For instance, in the developing rat cor- (Perego et al., 2011). Whether this relatively rapid ALLO’s action tex, GABA-A receptor activation leads to the opening of L-type on GABA-A receptor in glial cells may lead to ion channels open- voltage-gated calcium channels, resulting in an increase of cal- ing, as occur in neuronal cells, should not be totally excluded and cium influx, which in turn leads to phosphorylation and activation deserves further investigation. of the transcription factor cAMP response element-binding pro- tein (CREB) and finally controls neuronal synthase CONCLUSION and brain derived neurotrophic factor expression (Mantelas et al., In this review we focused our attention on the putative effects 2003). However, it should be underlined that the presence of of ALLO on the glial cells of the CNS and PNS, with a partic- endogenous GABA is the first precondition for ALLO’s allosteric ular emphasis on ALLO’s regulation of glial physiology in nor- action. The synthesis of GABA was previously shown in astrocytes mal and/or pathological conditions. The mechanism by which and oligodendrocytes, and very recently also in SC (Magnaghi ALLO exerts these controls is part of the “non-genomic” action et al., 2010). of steroids, involving the rapid modulation of neurotransmitter Therefore, the interaction among the neurosteroid ALLO, receptors such as the GABA receptors. However, ALLO’s action GABA-A, and GABA-B receptors is relevant, at least in the PNS, for on GABA-A receptor somehow involves downstream “genomic” the bidirectional cross-talk between neurons and SC (Figure 1). mechanisms that are yet to be completely elucidated. In SC these GABA, coming from the neuronal compartment, or produced intracellular mechanisms imply the activation of PK-A and PK-C by SC (Magnaghi et al., 2010) may affect the paracrine inter- signaling pathways, which in turn may determine the physio- play between these cells. In fact, data obtained with the specific logic control of SC proliferation/differentiation. Nevertheless, an GABA-B ligand baclofen suggest that extracellular GABA might involvement of other intracellular pathways, for instance ion cal- interact with GABA-B receptors on SC, decreasing their pro- cium channels,as observed in rat neuroprogenitor cells and human liferation, the cAMP levels and the myelin proteins expression. neural stem cells (Wanget al.,2005),should be further investigated. This effect promotes SC differentiation. ALLO produced by SC (Baulieu and Schumacher, 1997; Melcangi et al., 1999b) via a ACKNOWLEDGMENTS direct action on GABA-A receptor modulates the GABA-B recep- The authors are grateful to Dr. Ben Minogue, The University tor expression, distribution and responsiveness, and in turn its of Manchester, UK, for helpful discussion and reviewing of the desensitization. Contemporarily, ALLO may increase some myelin manuscript.

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Verdi, J. M., and Campagnoni, A. T. regulating polysialic acid-neural cell pharmacology of vertebrate GABAA conducted in the absence of any (1990). Translational regulation by adhesion molecule expression and receptor subtypes. Int. Rev. Neuro- commercial or financial relationships steroids. Identification of a steroid cell migration. J. Cell Biol. 135, biol. 38, 95–138. that could be construed as a potential modulatory element in the 5’- 1565–1581. Williamson, A. V., Mellor, J. R., Grant, conflict of interest. untranslated region of the myelin Wang, J. M., Johnston, P. B., Ball, A. L., and Randall, A. D. (1998). basic protein messenger RNA. J. Biol. B. G., and Brinton, R. D. (2005). Properties of GABA(A) receptors in Received: 30 July 2011; paper pending Chem. 265, 20314–20320. The neurosteroid allopregnanolone cultured rat oligodendrocyte prog- published: 23 September 2011; accepted: Vergnano, A. M., Schlichter, R., and promotes proliferation of rodent enitor cells. Neuropharmacology 37, 05 December 2011; published online: 28 Poisbeau, P. (2007). PKC activa- and human neural progenitor cells 859–873. December 2011. tion sets an upper limit to the and regulates cell-cycle gene and Yokoi, H., Tsuruo, Y., Miyamoto, Citation: Faroni A and Magnaghi functional plasticity of GABAergic protein expression. J. Neurosci. 25, T., and Ishimura, K. (1998). V (2011) The neurosteroid allo- transmission induced by endoge- 4706–4718. Steroid 5 alpha-reductase type 1 pregnanolone modulates specific nous neurosteroids. Eur. J. Neurosci. Whiting, P. J., Mcallister, G., Vassi- immunolocalized in the adrenal functions in central and peripheral 26, 1173–1182. latis, D., Bonnert, T. P., Heavens, gland of normal, gonadectomized, glial cells. Front. Endocrin. 2:103. doi: Von Blankenfeld, G., Trotter, J., and R. P., Smith, D. W., Hewson, L., and -supplemented 10.3389/fendo.2011.00103 Kettenmann, H. (1991). Expression O’Donnell, R., Rigby, M. R., Siri- rats. Histochem. Cell Biol. 109, This article was submitted to Frontiers and developmental regulation of a nathsinghji, D. J., Marshall, G., 127–134. in Neuroendocrine Science, a specialty of GABAA receptor in cultured murine Thompson, S. A., Wafford, K. A., Ziak, D., Chvatal, A., and Sykova, E. Frontiers in Endocrinology. cells of the oligodendrocyte lineage. and Vasilatis, D. (1997). Neuronally (1998). Glutamate-, kainate- and Copyright © 2011 Faroni and Magnaghi. Eur. J. Neurosci. 3, 310–316. restricted RNA splicing regulates the NMDA-evoked membrane currents This is an open-access article distributed Wang, C., Pralong, W. F., Schulz, expression of a novel GABAA recep- in identified glial cells in rat under the terms of the Creative Commons M. F., Rougon, G., Aubry, J. M., tor subunit conferring atypical func- spinal cord slice. Physiol. Res. 47, Attribution Non Commercial License, Pagliusi, S., Robert, A., and Kiss, tional properties. J. Neurosci. 17, 365–375. which permits non-commercial use, dis- J. Z. (1996). Functional N-methyl- 5027–5037. tribution, and reproduction in other D-aspartate receptors in O-2A glial Whiting, P. J., Mckernan, R. M., and Conflict of Interest Statement: The forums, provided the original authors and precursor cells: a critical role in Wafford, K. A. (1995). Structure and authors declare that the research was source are credited.

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