Elsevier

Brain Research

Volume 1040, Issues 1–2, 8 April 2005, Pages 29-35
Brain Research

Research report
Intracellular oxidation of allopregnanolone by human brain type 10 17beta-hydroxysteroid dehydrogenase

https://doi.org/10.1016/j.brainres.2005.01.022Get rights and content

Abstract

Allopregnanolone is a positive allosteric modulator of GABAA receptors, generated by the reduction of 5α-dihydroprogesterone (5α-DHP) in astrocytes. This neuroactive steroid can be inactivated by its 3α-oxidation to yield 5α-DHP. It was found that 5α-DHP levels in HEK293 cells expressing type 10 17β-hydroxysteroid dehydrogenase (17β-HSD10), but not its catalytic inactive mutant, increased significantly as allopregnanolone was added to culture media. The results demonstrate that mitochondrial 17β-HSD10 effectively catalyzes the intracellular oxidation of allopregnanolone. Moreover, brain astrocytes contain a moderate level of 17β-HSD10, which is elevated in activated astrocytes of brains with Alzheimer type pathology, including sporadic Alzheimer's disease (AD) and Down's syndrome with AD. The distribution of 17β-HSD10 was found not to parallel that of 3α-HSD3. Cerebral cortex has the lowest level of 17β-HSD10; whereas the hippocampus, hypothalamus, and amygdala possess relatively higher levels of this enzyme. The catalysis of 17β-HSD10 appears to be essential for maintaining normal functions of GABAergic neurons. The elevated level of 17β-HSD10 in activated astrocytes is a new feature found in brains of people with AD, and it may have important impact on AD pathogenesis.

Introduction

3α,5α-Tetrahydroprogesterone (allopregnanolone) is a natural, positive allosteric modulator of γ-aminobutyric acid type A (GABAA1) receptors [28]. The 3α-hydroxyl group of such neuroactive steroids is crucial for modulating GABAA receptors [27] by increasing the receptor opening frequency and duration [17], [28]. In addition, allopregnanolone may modulate GABAA receptor subunit composition to alter seizure susceptibility [40]. Among neurosteroids, allopregnanolone displays special clinical significance [32]. For example, this 3α,5α-tetrahydrosteroid can attenuate the endocrine response to stress [33] and plays a role in the suppression of seizures [10]. Allopregnanolone, which is generated by the reduction of 5α-dihydroprogesterone (5α-DHP) [14], [43], does not interact with intracellular steroid hormone receptors [8], [38]. However, it can modulate gene expression after intracellular oxidation into 5α-DHP [8], [38], [39]. Therefore, allopregnanolone exerts rapid, non-genomic effects as well as long-term, genomic effects [29], [39].

The 3α-oxidation of allopregnanolone was reportedly catalyzed by 3α-HSD3 [14], [43], 17β-hydroxysteroid dehydrogenase type 10 (HSD-10) [23], and microsomal 3α-HSDs [7]. Human 3α-HSD3 is a member of the aldo-keto reductase (AKR) family, and also known as AKR1C2 [37], [43]. Transfected 3α-HSD3 was recently reported to act intracellularly as a 3-ketosteroid reductase instead of an oxidase [37]. Although the catalytic efficiency of HSD-10 was reported to be two orders of magnitude greater than 3α-HSD3 [23], to establish its role in neurosteroid metabolism, it is necessary to determine whether human HSD-10 is able to competently catalyze the intracellular oxidation of allopregnanolone.

In this report, we demonstrate that mitochondrial HSD-10 effectively catalyzes the intracellular oxidation of allopregnanolone. We also show that human HSD-10 is differentially distributed in various brain regions, and this enzyme is especially abundant in activated astrocytes of brains with Alzheimer type pathology.

Section snippets

Materials

Pregnan-3α-ol-20-one, 5α-[9, 11, 12-3H(N)]- was purchased from Perkin-Elmer Life Sci., Inc. Allopregnanolone, 5α-DHP, mAb GFAP, and other standard biochemicals were purchased from Sigma. Total protein isolated from various human brain regions, known as Protein Medleys, ready for studying the tissue distribution of specific proteins [11], [21], was purchased from Clontech. Rabbit antibodies raised against human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Chemicon. Both

Oxidation of allopregnanolone by HEK 293 cells expressing 17β-HSD10

To determine whether HSD-10 could catalyze the oxidation of allopregnanolone in intact cells, we examined HEK 293 cells stably transfected with pCR3.1/17HSD10 and pCR3.1/17HSD10(Y168F). These two transfectants expressed wild-type HSD-10 and its mutant HSD-10(Y168F), respectively, to approximately equal levels [22]. Since 3α-oxidation of allopregnanolone generates 5α-DHP, accumulation of 5α-DHP in cells is indicative of the oxidation of allopregnanolone. The results were then standardized by the

Discussion

Human HSD-10 is a homotetrameric protein with a molecular mass of 108 kDa [18], [21]. This intriguing member of the SDR family was also known as brain short chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) [18], [20], [21], which is localized in the mitochondria [20], [21]. This localization was confirmed here again by immunocytochemistry (Figs. 3a–d). However, it is noteworthy that images of activated astrocytes immunostained with mAb 10E5 (Fig. 3l) are strikingly different from those of

Acknowledgments

We thank D. Miller for critically reading the manuscript. We also thank P. Mehta and R. Kascsak for providing antibodies as well as J. Wegiel for excellent technical assistance. This work was supported in part by New York State Office of Mental retardation and Developmental Disabilities.

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