Review
Neurosteroid biosynthesis in vertebrate brains

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Abstract

In mammals, neurosteroids are now known to be synthesized de novo in the brain as well as other areas of the nervous system through mechanisms at least partly independent of the peripheral steroidogenic glands. However, limited information is available on neurosteroids in non-mammalian vertebrates. We therefore have attempted to demonstrate neurosteroid biosynthesis in the brain of birds and amphibians. These vertebrate brains possessed the steroidogenic enzymes, cytochrome P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ54-isomerase (3β-HSD), and produced pregnenolone, pregnenolone sulfate ester and progesterone from cholesterol. Significant seasonal changes in neurosteroids in the brain were observed in seasonally breeding vertebrates. In addition, we attempted to identify the cell type involved in neurosteroidogenesis in mammalian and non-mammalian vertebrates in order to understand the physiological role of neurosteroids. Glial cells are generally accepted to be the primary site for neurosteroid formation, but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the brain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. This paper summarizes the advances made in our understanding of neurosteroid biosynthesis, including neuronal neurosteroidogenesis, in a variety of vertebrate types.

Introduction

In vertebrates, steroid hormones, supplied by the peripheral steroidogenic glands, regulate several important brain neuronal functions during development which persist into adulthood. Peripheral steroid hormones cross the blood–brain barriers, due to their lipid solubility, and act on brain tissues through intracellular receptor-mediated mechanisms that regulate the transcription of specific genes [20], [40]. Gonadal androgens, for instance, act on the brain to influence several reproductive behaviors in vertebrates. Many of the brain regions that control a variety of reproductive behaviors contain a high proportion of cells that concentrate androgenic hormones. Therefore, the brain is considered to be a target site of peripheral steroids. In addition to direct steroidal actions, the metabolism of peripheral steroids in brain tissues can result in biotransformation and the production of biologically active metabolites. Indeed, androgenic action in the vertebrate brain is often mediated by the enzymatic activity of cytochrome P450arom which catalyzes the conversion of androgen to estrogen. Both P450arom and estrogen receptors are expressed in several brain regions, including the hypothalamus and preoptic area, which are involved in the control of reproductive behaviors.

On the other hand, new findings have been obtained that the nervous system itself may form steroids de novo. The pioneering discovery of Baulieu and his colleagues, using rodents, has opened the door of a new research field for many laboratories. Pregnenolone and dehydroepiandrosterone, as unconjugated steroids, and their fatty acid or sulfate esters, accumulate within the brains of several mammalian species [13], [14], [25], [32], [39], [52], [53], [54]. The brain content of these steroids remains constant even after the removal of peripheral steroids by procedures such as adrenalectomy, castration and hypophysectomy. This suggests that the brain can synthesize steroids de novo [13], [14], [25], [52], [53], [54]. Such steroids synthesized in the brain are called neurosteroids.

In contrast to extensive mammalian studies, especially using rodents, little has been known regarding the formation of neurosteroids in the brain of non-mammalian vertebrates. We therefore looked for neurosteroids formed from cholesterol in the brains of both avian [61], [62], [63], [67] and amphibian species [60]. Independently, other groups, such as Vaudry’s laboratory [42] and Schlinger’s laboratory [70], also contributed to this area. The formation of several neurosteroids is now known to occur in non-mammalian vertebrates. Our recent studies have focused on investigating physiological changes in neurosteroids in the brain of seasonally breeding vertebrates [10], [60], as this would contribute to our understanding of neurosteroid action.

In addition to understand the physiological role of neurosteroids in brain function, it is also essential to identify the cells involved in neurosteroidogenesis. It is generally accepted that in mammals, glial cells play a major role in neurosteroid formation and metabolism in the brain and both oligodendrocytes and astrocytes are the primary site for pregnenolone synthesis, an initial step of neurosteroidogenesis. However, whether neurons located in the brain produce neurosteroids has remained unclear. With these findings as a background, we have demonstrated the presence and activity of steroidogenic enzymes in a cerebellar neuron. Interestingly, the Purkinje cell possesses steroidogenic enzymes and produces neurosteroids de novo in a variety of vertebrates including mammalian species [60], [62], [63], [64], [65], [67]. This is the first demonstration of neuronal de novo neurosteroidogenesis in the brain and serves an excellent model for the study of physiological roles of neurosteroids in the brain.

In this review, we summarize the findings of neurosteroid biosynthesis in the brain of several vertebrates obtained by our recent studies and the related studies of other laboratories. These also include observations on neuronal neurosteroidogenesis in vertebrate brains and physiological changes in neurosteroids in the brains of seasonal breeders. For detailed information of neurosteroids in rodents the reader is referred to an excellent review [7].

Section snippets

Vertebrate brains produce neurosteroids

Pregnenolone, a 3β-hydroxy-Δ5-steroid, is a main precursor of steroid hormones secreted by steroidogenic glandular cells. The formation of pregnenolone is initiated by the cleavage of the cholesterol side-chain by cytochrome P450scc, a rate-limiting mitochondrial enzyme originally found in peripheral steroidogenic glands. Therefore, it is essential to demonstrate the formation of pregnenolone in the brain. A number of studies with several species of mammals have reported that the brain contains

Age- and region-specific expressions of neurosteroidogenic enzymes in the brain

To clarify the biosynthetic and metabolic pathways of neurosteroids in the brain, extensive studies with several vertebrates, especially mammals, have been carried out by many laboratories. As indicated above, the presence of cytochrome P450scc and 3β-HSD has been well established in the vertebrate brain, whereas limited information has been available for the enzyme 17α-hydroxylase/c17,20-lyase (cytochrome P45017α,lyase), which converts pregnenolone to dehydroepiandrosterone, one of the most

Identification of neurosteroidogenic cells in the brain

To determine the precise localization of the steroidogenic enzyme P450scc in the brain, immunohistochemical analysis with the antibody against P450scc has been conducted in several vertebrate species. In the first immunohistochemical description of cytochrome P450scc by Le Goascogne et al. [33], an intense immunoreaction was detected in the white matter zone throughout the rat brain. The biochemical study in the rat further demonstrated that oligodendrocyte mitochondria convert cholesterol to

Physiological changes in neurosteroids in the brain of seasonal breeders

As mentioned above, de novo steroidogenesis from cholesterol appears in the brain of several vertebrates, such as mammals, birds and amphibians. Physiological changes in neurosteroid levels must be taken into account when understanding the function of neurosteroids in the vertebrate brain. If neurosteroids are involved in some important brain functions, it would be expected that they would change under different physiological conditions. To test this hypothesis, wild animal species may serve as

Neurosteroid actions in the brain

Future directions of neurosteroid studies should establish the mode of action of neurosteroids and their physiological function in the brain. Several investigators have reported that in mammals, neurosteroids mediate their actions through ion-gated channel receptors, such as GABAA and N-methyl-d-aspartate [30], [31], [34], [35], [36], [45], [50], [58], [72]. For example, pregnenolone and its sulfate ester are thought to act as an agonist and antagonist of GABAergic neurotransmission [35], [37],

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (08454265, 10874129 and 11170237 to K.T.) and by the International Joint Research Project of the British Council. We are grateful to Drs S. Kominami, T. Yamazaki, Y. Furukawa, and M. Usui (Hiroshima University, Higashi-Hiroshima, Japan) for their collaboration.

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