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Abstract

It is now clearly established that steroids can be synthesized de novo by the vertebrate brain. Such steroids are called neurosteroids. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. In the middle 1990s, the Purkinje cell, an important cerebellar neuron, was identified as a major site for neurosteroid formation in vertebrates. This discovery has allowed deeper insights into neuronal neurosteroidogenesis and biological actions of neurosteroids have become clear by the studies using the Purkinje cell as an excellent cellular model, which is known to play an important role in memory and learning processes. From the past 10 years of research on mammals, we now know that the Purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such neurosteroid actions that may be mediated by neurotrophic factors contribute to the formation of cerebellar neuronal circuit during neonatal life. Allopregnanolone, a progesterone metabolite, is also synthesized in the cerebellum and acts on Purkinje cell survival in the neonate. The aim of this review is to summarize the current knowledge regarding the biosynthesis and biological actions of neurosteroids in the Purkinje cell during development.

Keywords

neurosteroids progesterone estradiol neuronal growth synaptogenesis Purkinje cell

Introduction

It has long been thought that steroidogenic glands including the adrenal cortex, gonads and placenta (only in mammals), are the only sources of steroid hormones in vertebrates. These peripheral steroid hormones cross the blood-brain barrier, owing to their lipophilic nature, and act on brain tissues through intracellular receptor-mediated mechanisms that regulate several important brain neuronal functions during development which persist into adulthood in vertebrates. Accordingly, the brain has traditionally been considered to be a target site of peripheral steroid hormones. By contrast, new findings over the past twenty years have shown that the brain itself also has the capability of forming steroids de novo from cholesterol, the so-called “neurosteroids.”^{1

5} The brain content of neurosteroids remains constant even after the removal of peripheral steroid hormones by procedures such as adrenalectomy, castration, and hypophysectomy.^{6

8} The formation of neurosteroids in the brain was originally demonstrated in mammals by the pioneering studies of Baulieu and his colleagues^6,7,9–16 and subsequently in other vertebrates, such as birds,^8,17–29 amphibians^{30

37} and fish³⁸ by the independent studies of several laboratories of Tsutsui, Vaudry, Schlinger, etc.^{1

5} Thus, de novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. The discovery of neurosteroids has opened the door to a new research field in “neuroscience.”

The identification of neurosteroidogenic cells is essential to analyze neurosteroid actions in brain function. It is also necessary to know which neurosteroids are synthesized in specific brain regions at specific period during development. Such information enables to develop hypotheses predicting the potential roles of those neurosteroids in the developing and adult brains. Thus, the studies for this exciting new area in “neuroscience” should be focused on the biosynthesis and biological actions of neurosteroids produced locally in the identified neurosteroidogenic cells underlying important brain functions. The oligodendrocyte was first accepted to be the primary site for neurosteroid formation in the brain.^1,3 Subsequently astrocytes¹⁵ and some neurons¹⁶ were shown to express some steroidogenic enzymes. Thus, glial cells were generally accepted to be the site for neurosteroid formation. However, whether neurons located in the brain produce neurosteroids was, until recently, unknown in vertebrates.

In the middle 1990s, Tsutsui and co-workers discovered that the Purkinje cell, an important cerebellar neuron, is a major site for neurosteroid formation in a variety of vertebrates including mammals.^{8,17,24,34,38–43} This was the first demonstration of de novo neuronal neurosteroidogenesis in the brain. This discovery has provided the opportunity to understand neuronal neurosteroidogenesis in the brain. In mammals, the Purkinje cell possesses several kinds of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase (3β-HSD), and actively produces progesterone during neonatal life^39,40,44 (Fig. 1). Allopregnanolone (3α,5α-tetrahydroprogesterone), a progesterone metabolite, is also synthesized in the neonatal cerebellum^{42,43,45–48} (Fig. 1). Subsequently, biological actions of progesterone^{49

52} and the progesterone metabolite allopregnanolone⁵³ have become clear by the studies on mammals using the Purkinje cell which is known to play an essential role in the process of memory and learning. Furthermore, this neuron expresses a key enzyme of estrogen formation, cytochrome P450 aromatase (P450arom), and produces estradiol in the neonate^41,46 (Fig. 1). Estradiol also contributes to important events in the developing Purkinje cell.^41,54

Figure 1.

Neurosteroid formation in the Purkinje cell. The Purkinje cell is a major site for neurosteroid formation in the brain. The Purkinje cell possesses several kinds of steroidogenic enzymes. The expression of P450scc remains during neonatal development and in adulthood, indicating the constant production of pregnenolone. This neuron also produces actively progesterone due to an increase of 3β-HSD activity only during neonatal life. Allopregnanolone is also metabolized by the enzymes 5α-reductase and 3α-HSD from progesterone during neonatal life. Estrogen formation in the Purkinje cell also occurs in the neonate because this neuron further expresses P450_17α,lyase and P450arom. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase; P450_17α,lyase cytochrome P450 17α-hydroxylase/c17,20-lyase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; 3α-HSD, 3α-hydroxysteroid dehydrogenase; P450arom, cytochrome P450 aromatase.

Thus, the discovery of the Purkinje cell as a major site for neurosteroid formation has forced us to re-evaluate our understanding about the mechanisms regulating neuronal development and functions in the brain. This review summarizes the advances made in our understanding of the biosynthesis and biological actions of neurosteroids in the Purkinje cell. Based on new findings obtained by the studies using this important neuron, this review also describes what are currently known about the mode of action and functional significance of neurosteroids. For detailed information of neurosteroids in glial cells the reader is referred to other reviews.^1,3

Discovery of the Purkinje Cell as a Major Site for Neurosteroidogenesis

The new concept of de novo neurosteroid formation from cholesterol in the Purkinje cell, an important cerebellar neuron, derived from the observations of Tsutsui and co-workers. Pregnenolone, a 3β-hydroxy-Δ⁵-steroid, is a main precursor of steroid hormones secreted by peripheral steroidogenic glands, such as gonads and adrenals. The formation of pregnenolone is initiated by the cleavage of the cholesterol side-chain by P450scc, a rate-limiting mitochondrial enzyme originally found in peripheral steroidogenic glandular cells. Therefore, it is essential to demonstrate the presence of P450scc in the Purkinje cell. The first immunohistochemical study in quail using an antibody against P450scc reported that the striking observation of the distribution of intense immunoreactive cells in the cerebellar cortex.^8,17 The distribution of immunoreactive cell bodies and fibers was coincident with the location of somata and dendrites of Purkinje cells.^8,17 Western immunoblot analysis confirmed the presence of P450scc in Purkinje cells.^8,17 These avian findings provided the first evidence for the neuronal location of P450scc.

Subsequently, Tsutsui and co-workers extended these findings and investigated the presence of P450scc in rat Purkinje cells.³⁹ An antibody against inositol triphosphate (IP₃) receptor, a marker of the Purkinje cell, recognized P450scc-immunoreactive cerebellar cells that showed no immunoreaction with glial fibrillary acidic protein (GFAP), a specific marker of astrocytes.³⁹ Thus, immunoreaction with P450scc antibody was confined to the somata and dendrites of Purkinje cells in the rat cerebellum.³⁹ Interestingly, P450scc appeared in the rat Purkinje cell immediately after its differentiation, and the expression of this enzyme persisted during neonatal development into adulthood.³⁹ In addition to higher vertebrates, Tsutsui and co-workers further identified P450scc in the Purkinje cell of amphibians.³⁴ Taken together, these findings obtained in both higher and lower vertebrates indicate that Purkinje cells possess P450scc and produce pregnenolone (Fig. 1). Steroidogenic acute regulatory protein (StAR) was also found in Purkinje cells⁴⁴ (Fig. 1). StAR is involved in the transport of cholesterol to the inner mitochondrial membrane, in which P450scc is localized, and thus plays a key role in acute steroid biosynthesis in peripheral steroidogenic glands.⁵⁵ Therefore, StAR may also contribute to the regulation of pregnenolone formation in the Purkinje cell.

After the discovery of pregnenolone formation in the Purkinje cell, we now know that this neuron is a major site of neurosteroid formation in a variety of vertebrates. From the past ten years of research on mammals, the colocalization of several kinds of steroidogenic enzymes in the Purkinje cell has been demonstrated as described below.

Biosynthesis of Neurosteroids in the Purkinje Cell

Progesterone formation and metabolism in the Purkinje cell

Progesterone is known to be a sex steroid hormone and acts on brain tissues through nuclear progesterone receptors. In addition to this classical concept, Tsutsui and co-workers demonstrated de novo progesterone formation from cholesterol in the Purkinje cell in the late 1990s. Because the biosynthesis of progesterone from pregnenolone is performed by 3β-HSD, a membrane-bound mitochondrial enzyme, the demonstration of the expression of 3β-HSD in the Purkinje cell is therefore essential to establish the concept of de novo progesterone formation from cholesterol in this neuron. Tsutsui and co-workers found that Purkinje cells express not only P450scc but also 3β-HSD (Fig. 1). RT-PCR and biochemical analyses showed the expression of 3β-HSD and its enzymatic activity in the rat cerebellum.⁴⁰ Using in situ hybridization of 3β-HSD mRNA, the site of 3β-HSD expression was localized in Purkinje cells and external granule cells.⁴⁰ Thus, both P450scc and 3β-HSD are expressed in Purkinje cells (Fig. 1). The colocalization of P450scc and 3β-HSD in external granule cells is still unclear. The expression of 3β-HSD in Purkinje cells was also evident in other vertebrates.^2,38 Surprisingly, the expression of 3β-HSD increased during neonatal life, unlike P450scc.^39,40 Such an age-dependent expression of 3β-HSD was confirmed by biochemical studies together with HPLC analysis, indicating an increase of progesterone formation during neonatal life.⁴⁰ Thus, this neuron actively produces progesterone as a product of an increase of 3β-HSD activity during neonatal life⁴⁰ (Fig. 1).

Biochemical analysis together with HPLC and TLC further revealed that the progesterone metabolite allopregnanolone is also found in the cerebellum during neonatal life.^29,45,46,48 The expression of 5α-reductase and 3α-HSD that metabolize progesterone to allopregnanolone has been found in the Purkinje cell^42,43(Fig. 1).

Estrogen formation in the Purkinje cell

Estradiol is also known to be a sex steroid and acts on brain tissues. P450arom is a key enzyme of estrogen formation in peripheral steroidogenic glands. Recently, Tsutsui and co-workers have further demonstrated the expression of P450arom in Purkinje cells during neonatal life⁴¹ (Fig. 1). RT-PCR and in situ hybridization analyses showed that the expression of P450arom mRNA in the cerebellum is restricted to Purkinje cells and external granule cells in neonatal rats.⁴¹ A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate are higher than those in prepubertal and adult rats.⁴¹ In addition, a recent study has shown the expression and activity of cytochrome P450 17α-hydroxylase/c17,20-lyase (P450_17α,lyase), which converts pregnenolone to dehydroepiandrosterone (DHEA) or progesterone to androstenedione, an immediate precursor of estrogen formed by P450arom, in the Purkinje cell²⁴ (Fig. 1). These studies indicate estrogen formation in the Purkinje cell during neonatal life (Fig. 1).

Biological Actions of Neurosteroids in the Purkinje Cell

Promotion of Purkinje dendritic growth, spinogenesis and synaptogenesis by progesterone

Because the Purkinje cell is a major site for progesterone formation and metabolism in the brain, this neuron has served as an excellent cellular model for the study of biological actions of these neurosteroids in mammals. Purkinje cells actively synthesize progesterone during the neonatal period, as the expression of 3β-HSD and its enzymatic activity increase in neonatal rats⁴⁰ (Fig. 1). The progesterone metabolite allopregnanolone is also synthesized in the cerebellum of neonatal rats.^{45

48} It is well known that in the rat marked morphological changes occur in the cerebellum after birth during neonatal life and the formation of the cerebellar cortex becomes complete in the neonate through the processes of migration of external granule cells, neuronal and glial growth, and synaptogenesis.^56,57 According to Altman and Bayer,⁵⁸ the rat Purkinje cell is prenatally formed and its maturation starts immediately after birth. Thus, cerebellar development is dramatic during neonatal life, when cerebellar progesterone and allopregnanolone concentrations are high.^40,45,46 Therefore, progesterone and/or allopregnanolone may be involved in the formation of the cerebellar neuronal circuit by promoting neuronal growth and neuronal synaptic contact.

To test this hypothesis, Tsutsui and co-workers examined the effects of progesterone and allopregnanolone, produced as neurosteroids in the Purkinje cell during neonatal life, on neuronal growth, spinogenesis, and synaptogenesis in the rat cerebellum. In vitro studies using cultured cerebellar slices of newborn rats showed that progesterone promotes dendritic growth and dendritic spine formation of the Purkinje cell^49,50 (Fig. 2). A similar result was obtained by in vivo studies.^49,50 The stimulatory action of progesterone on Purkinje cell dendrites was completely blocked by the progesterone receptor antagonist mifepristone (RU486) in vitro by combined administration of progesterone and RU 486.^49,50 Furthermore, in vivo administration of RU 486 during the endogenous peak of progesterone inhibited dendritic growth and dendritic spine formation of the Purkinje cell.^49,50 Electron micro-scopic analysis further revealed that progesterone induces an increase of the density of dendritic axospinous synapses on the Purkinje cell^49,50 (Fig. 2). In contrast, there was no significant change in the density of dendritic shaft synapses after progesterone administration.^49,50 The effect of progesterone on Purkinje dendritic spine synapses was also blocked by RU486.^49,50 In contrast to progesterone, there was no significant effect of allopregnanolone on these aspects of Purkinje development.^49,50 These results indicate that progesterone promotes the dendritic growth, spinogenesis, and synaptogenesis of Purkinje cells during cerebellar development (Fig. 2).

Figure 2.

The proposed mechanisms of action of progesterone in the Purkinje cell during development. Progesterone acts on the Purkinje cell through intranuclear receptor (PR)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Thus, progesterone produced in the Purkinje cell may mediate its actions through an “intracrine” mechanism. Progesterone may induce the expression of some neurotrophic factors that directly promote Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life. On the other hand, progesterone may also act on Purkinje cells through the mechanisms mediated by 25-Dx, which is associated with membrane structures of the endoplasmic reticulum and Golgi apparatus. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase; PR, progesterone receptor; PRE, progesterone response element; Golgi, Golgi apparatus; ER, endoplasmic reticulum.

Neuroprotection of Purkinje cells by progesterone and allopregnanolone

In addition to organizing actions of progesterone as described above, it has been reported that the anti-progesterone RU486 protects Purkinje cells from developmental cell death, although progesterone does not possess any effect on Purkinje cell survival.⁵¹ This protective effect of RU486 is considered to be independent on the activation of nuclear progesterone receptor (PR).⁵¹

Purkinje cells metabolize some of progesterone to allopregnanolone during neonatal life.^{45

48} Although allopregnanolone failed to promote the dendritic growth, spinogenesis, and synaptogenesis of Purkinje cells,^49,50 it has been shown that allopregnanolone is involved in Purkinje and granule cell survival.⁵³ The Niemann-Pick type C (NP-C) mouse has been used as an excellent animal model for understanding the action of allopregnanolone. NP-C is an autosomal recessive, childhood neurodegenerative disease characterized by defective intracellular cholesterol trafficking, resulting in Purkinje cell degeneration as well as neuronal degeneration in other regions. Brains from adult NP-C mice contained less allopregnanolone than wild-type brain.⁵³ Administration of allopregnanolone to neonatal NP-C mice increased Purkinje cell survival and delayed neurodegeneration.⁵³

Promotion of Purkinje dendritic growth, spinogenesis and synaptogenesis by estradiol

As mentioned above, Purkinje cells highly express P450arom in the neonate, indicating its age-dependent expression. In addition, estradiol levels in the neonate are higher in the cerebellum than in the plasma.⁴¹ To clarify the action of estradiol in Purkinje cells during neonatal life, Tsutsui and co-workers analyzed the effect of estradiol on dendritic growth of Purkinje cells by both in vitro and in vivo studies using newborn rats.⁴¹ Treatment of cerebellar cultures with estradiol promoted the dendritic growth of Purkinje cells in a dose-dependent manner with active doses being in the range of physiological levels of estradiol measured in the cerebellum.⁴¹ A similar morphological effect was also obtained by the in vivo treatment with estradiol.⁴¹ In contrast, the blockage of action of endogenous estrogen by treatment with tamoxifen, an estrogen receptor antagonist, had reversed effects on the Purkinje cell morphology. Further, estradiol treatment increased the densities of Purkinje dendritic spines⁴¹ and spine synapses.⁵⁴ These effects were also inhibited by tamoxifen.^41,54 Thus, estradiol also promotes the dendritic growth, spinogenesis, and synaptogenesis of Purkinje cells (Fig. 3).

Figure 3.

The proposed mechanisms of action of estradiol in the Purkinje cell during development. Estradiol acts on the Purkinje cell through intranuclear receptor (ERβ)-mediated mechanisms that promote dendritic growth, spinogenesis, and synaptogenesis in this neuron by genomic mechanisms. Both Purkinje cells and granule cells express BDNF and TrkB, a receptor for BDNF. Estradiol induces the expression of BDNF, which may act on Purkinje cells and granule cells through TrkB-mediated mechanisms to promote Purkinje dendritic growth, spinogenesis, and synaptogenesis. StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450 side-chain cleavage enzyme; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴-isomerase; P450_17α,lyase cytochrome P450 17α-hydroxylase/c17,20-lyase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; P450arom, cytochrome P450 aromatase; ERβ, estrogen receptor-β; ERE, estrogen response element; BDNF, brain-derived neurotrophic factor; TrkB, BDNF receptor.

Mode of Action and Functional Significance of Neurosteroids in the Purkinje Cell

Mode of action and functional significance of progesterone

To understand the mode of action of progesterone, the expression of PR in the cerebellum was then characterized in neonatal rats. Interestingly, intranuclear PR-A and PR-B were expressed in the Purkinje cell.^49,50,52 It is therefore considered that progesterone acts directly on Purkinje cells through intranuclear receptor-mediated mechanisms to promote Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal development^49,50,52 (Fig. 2). Such genomic actions of progesterone may be essential for the formation of the cerebellar neuronal circuit. Thus, progesterone produced in the Purkinje cell may mediate its actions through an “intracrine” mechanism.

On the other hand, Purkinje cells express the putative membrane progesterone receptor, 25-Dx, during neonatal life.⁵⁹ RT-PCR and Western immunoblot analyses revealed the expressions of 25-Dx and its mRNA in the rat cerebellum, which increased during neonatal life.⁵⁹ By immunocytochemistry, the expression of 25-Dx was localized in the Purkinje cell and external granule cell layer.⁵⁹ At the ultrastructural level, 25-Dx immunoreactivity was associated with membrane structures of the endoplasmic reticulum and Golgi apparatus in the Purkinje cell.⁵⁹ It is possible that progesterone may promote dendritic growth, spinogenesis, and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate⁶⁰ (Fig. 2). This protein is now named ‘progesterone receptor membrane component 1’ (PGRMC1), and there is now strong evidence that PGRMC1 mediates the anti-apoptotic actions of progesterone in both rat granulose and luteal cells and associates with another membrane protein, such as plasminogen activator inhibitor RNA-binding protein-1 (PAIRBP1).^{61

64} Future study is needed to demonstrate whether the promotion of Purkinje dendritic growth, spinogenesis, and synaptogenesis by progesterone is due to both genomic and nongenomic actions.

A series of the studies of Tsutsui and co-workers indicate that progesterone promotes Purkinje dendritic growth, spinogenesis, and synaptogenesis. Such organizing actions may contribute to the formation of the cerebellar neuronal circuit during neonatal life. Neurotrophins are attractive candidate regulators of Purkinje dendrite and spine development. It has been reported that neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are highly expressed in the developing cerebellum and are critical for proper development of Purkinje cells and granule cells.^{65

69} Therefore, progesterone may induce the expression of some neurotrophic factors that directly promote Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life.^70,71

In addition to the central nervous system, progesterone has also been shown to promote myelination in the peripheral nervous system via nuclear PR.^{72

74}

Mode of action and functional significance of estradiol

It has been reported that in the neonatal rat, Purkinje cells express estrogen receptor-β (ERβ).^75,76 Therefore, it is likely that estradiol acts directly on Purkinje cells through intranuclear ERβ-mediated mechanisms to promote dendritic growth, spinogenesis, and synaptogenesis in Purkinje cells during neonatal development (Fig. 3). This hypothesis was confirmed by the finding with the ER antagonist tamoxifen, which inhibited the effects of estrogen on Purkinje dendritic growth, spinogenesis, and synaptogenesis.^41,54 It is known that this anti-estrogen binds to ERs (ERα and ERβ) and activates transcription via activating protein-1 response elements⁷⁷ but blocks transcriptional activation through the classical estrogen response element and not producing any agonist effect via this pathway.^78,79 Thus, it is considered that the anti-estrogen tamoxifen blocks transcriptional activation of ERβ in the developing Purkinje cell. On the other hand, granule cells also express ERβ^75,76 (Fig. 3). Involvement of ERβ in the brain function has also been reported in the rat hypothalamus.^80,81

While ERβ appears to mediate effects of estradiol in Purkinje cell function, other receptors may also mediate effects of estradiol in other brain regions, such as hippocampus^{82

87} and hypothalamus.^88,89 The effect of estradiol on hippocampal CA1 pyramidal cell dendrite spine density requires the activation of N-methyl-D-aspartate (NMDA) receptors in adult female rats.⁸⁵ Such nongenomic estrogen actions may lead to alterations in gene expression. Hence, NMDA receptors may also mediate estradiol action in Purkinje cells. Future study is needed to demonstrate whether the promotion of Purkinje dendritic growth, spinogenesis, and synaptogenesis by estradiol is due to both genomic and nongenomic actions.

To demonstrate the functional significance of estradiol in the Purkinje cell during neonatal life, Tsutsui and co-workers further investigated estrogen actions on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using P450arom knock-out (ArKO) mice.⁵⁴ ArKO mice used in the study lack exons 1 and 2 and the proximal promoter region of the P450arom gene cyp19 (cytochrome P450, family 19).⁹⁰ Estradiol deficiency in ArKO mice decreased dendritic growth, spinogenesis, and synaptogenesis in Purkinje cells in the neonate.⁵⁴ In addition, administration of estradiol to ArKO mice increased Purkinje dendritic growth, spinogenesis, and synaptogenesis.⁵⁴ These results indicate physiological actions of endogenous estrogen on the promotion of dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell during neonatal development.

To elucidate the mode of action of estradiol, Tsutsui and co-workers further examined the expression of BDNF and NT-3 in response to estrogen actions in the neonate,⁵⁴ because these neurotrophic factors are known to be critical for proper development of Purkinje cells.^{65

69} Estrogen administration to neonatal wild-type (WT) mice or ArKO mice increased the BDNF level in the cerebellum, whereas the anti-estrogen tamoxifen decreased the BDNF level in WT mice similar to ArKO mice.⁵⁴ BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth.⁵⁴ In contrast to BDNF, estrogen administration did not influence the level of NT-3 in the cerebellum.⁵⁴ The NT-3 level also did not change in ArKO mice.⁵⁴ These results indicate that BDNF mediates estrogen action on the promotion of dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell during neonatal development (Fig. 3). In fact, the gene encoding BDNF contains a sequence similar to the canonical estrogen response element found in estrogen-target genes.⁹¹ In addition, BDNF increases levels of synaptic vesicle proteins, such as synaptophysin and synapsin 1, which are reliable markers of synaptogenesis, in the spinal neurons.⁹² Estrogen increases presynaptic and postsynaptic proteins, such as syntaxin, synaptophysin, and spinophilin, in the CA1 region of the primate hippocampus.⁹³ Furthermore, it has been reported that estrogen treatment induces these synaptic proteins in the CA1 region of hippocampus, and this effect is blocked by CI628, an anti-estrogen of the tamoxifen type.⁹⁴ The expression of P450arom mRNA in the cerebellum is restricted to Purkinje cells and external granule cells in the neonatal rats.^41,95–97 Both Purkinje cells and granule cells express BDNF^98,99 and TrkB, a receptor for BDNF^{100

102} (Fig. 3). It is therefore likely that estrogen induces the expression of BDNF, which acts on Purkinje cells and granule cells through TrkB-mediated mechanisms to promote Purkinje dendritic growth, spinogenesis, and synaptogenesis during neonatal life^54,71 (Fig. 3).

Neuroprotective and neurotrophic actions of estrogen have been reported by the studies using ArKO mice.^103,104 Neuroprotective effects of estrogen on dentate gyrus neurons in the hippocampus were mediated by estrogen-induced insulin-like growth factor-1 (IGF-1),¹⁰³ similar to neurotrophic effects of estrogen on Purkinje cells mediated by estrogen-induced BDNF.⁵⁴

Related Findings

Purkinje cell is a major site for neurosteroid formation and metabolism. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Allopregnanolone, a progesterone metabolite, acts on Purkinje cell survival in the neonate. In addition to Purkinje cells, the localization of neurosteroidogenic enzymes in other brain neurons has further characterized.^105,106 In the rat hippocampus, the localization of P450scc, P450_17α,lyase, and P450arom has been found in pyramidal neurons in the CA1-CA3 regions as well as granule cells in the dentate gyrus.^105,106 Thus, neurons as well as glial cells are now considered to play a major role in neurosteroid formation and metabolism in the brain. It has also been reported that estradiol promotes synaptogenesis in the hippocampus.^{82

87}

Progesterone and allopregnanolone have trophic effects and may improve cognitive function in mammals.^107,108 Estradiol is also implicated in the cognitive processes of the mammalian brain.^{109

112} Several regions of the mammalian brain involved in memory and cognition, such as the hippocampus, amygdala, cerebral cortex and cerebellum, are rich in estrogen receptors.^41,48,113 Thus, it is considered that progesterone, allopregnanolone and estradiol alter cognitive processes mediated by the hippocampus, cerebral cortex and cerebellum.

In addition to progesterone, allopregnanolone and estradiol, DHEA is also an abundant neurosteroid in the mammalian brain.^6,7,13 Compagnone and Mellon¹¹⁴ reported a similar action of DHEA and its sulfate ester (DHEAS) on neuronal growth using primary cultures of mouse embryonic neocortical neurons. According to Compagnone and Mellon,¹¹⁴ DHEA selectively increased the length of axons and the incidence of varicosities and basket-like process formations in vitro, whereas DHEAS selectively promoted branching and dendritic growth in vitro. Therefore, neurosteroids may play an important role in cortical organization in both the mammalian cerebellum and cerebrum during development. In contrast to progesterone and estradiol, DHEA and DHEAS may exert their organizing actions via nonclassical steroid hormone receptors in the mouse embryonic neocortical neuron.^114,115

Conclusions

Purkinje cell, an important cerebellar neuron, is a major site for neurosteroid formation in the brain. This neuron actively synthesizes progesterone and allopregnanolone, a progesterone metabolite, de novo from cholesterol during neonatal life when cerebellar neuronal circuit formation occurs. This neuron also produces estradiol in the neonate. Both progesterone and estradiol promote Purkinje dendritic growth, spinogenesis, and synaptogenesis. Such organizing actions that may be mediated by neurotrophic factors, such as BDNF, contribute to the formation of cerebellar neuronal circuit during neonatal life. The progesterone metabolite allopregnanolone is also involved in Purkinje and granule cell survival. Thus, the discovery of neurosteroid formation in the Purkinje cell has forced us to re-evaluate our understanding about the mechanisms regulating neuronal development and functions in the brain. Future directions for this exciting area of research should focus on physiological roles of neurosteroids, because Purkinje cells play an important role in the process of memory and learning. It is known that progesterone, allopregnanolone and estradiol influence cognitive processes, in particular memory and learning, in several brain regions including the cerebellum.^{107

113} Therefore, behavioral studies using neurosteroidogenic enzyme knock-out animals and electrophysiological studies on the occurrence of long-term potentiation and/or long-term depression are needed.

Conflict of Interest Statement

Grant support: Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (15207007, 16086206, and 18107002 to K.T.).

Disclosure Statement

The author has nothing to disclose.

Footnotes

Acknowledgments

I thank Hirotaka Sakamoto, Mariko Usui, Hanako Shikimi, Katsunori Sasahara, Shogo Haraguchi, Minoru Takase and Kazuyoshi Ukena for their work cited in this manuscript.

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Neurosteroid Biosynthesis and Action in the Purkinje Cell

Abstract

Keywords

Introduction

Discovery of the Purkinje Cell as a Major Site for Neurosteroidogenesis

Biosynthesis of Neurosteroids in the Purkinje Cell

Progesterone formation and metabolism in the Purkinje cell

Estrogen formation in the Purkinje cell

Biological Actions of Neurosteroids in the Purkinje Cell

Promotion of Purkinje dendritic growth, spinogenesis and synaptogenesis by progesterone

Neuroprotection of Purkinje cells by progesterone and allopregnanolone

Promotion of Purkinje dendritic growth, spinogenesis and synaptogenesis by estradiol

Mode of Action and Functional Significance of Neurosteroids in the Purkinje Cell

Mode of action and functional significance of progesterone

Mode of action and functional significance of estradiol

Related Findings

Conclusions

Conflict of Interest Statement

Disclosure Statement

Footnotes

Acknowledgments

References