Endogenous and synthetic neurosteroids in treatment of Niemann–Pick Type C disease

Over the past 20 years, genetic manipulation has revolutionised our understanding of male reproductive development and function. The advent of transgenic mouse lines has permitted elegant dissection of previously intractable issues. The development of the Cre/Lox system, which has permitted spatial and temporal localisation of genetic manipulation, has expanded upon this, and now makes up one of the primary approaches underpinning our increasing understanding of testis development and function. The success of conditional gene targeting is largely reliant upon the choice of Cre recombinase expressing mouse line, which is required to specifically target the correct cell type at the correct time. Presupposition that Cre lines will behave as expected has been one of the main oversights in the design of Cre/Lox experiments, as in practice, many Cre lines are prone to ectopic expression (both temporal and spatial), transgene silencing or genetic background effects. Empirical validation of the spatiotemporal profile of Cre expression prior to undertaking conditional gene targeting studies is essential and can be achieved through a combination of molecular and immunohistochemical approaches, along with in vivo examination of reporter gene expression in targeted tissues. This paper details the key considerations associated with exploitation of the Cre/Lox system and highlights a variety of validated Cre lines that have utility for conditional gene targeting within the testis.

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Good planning and serendipity: exploiting the Cre/Lox system in the testis

The steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme and specific 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerases initiate the essential process of steroidogenesis in the gonads. Testicular and ovarian expression of their respective genes, STARD1, CYP11A1 and gonadal HSD3B, is primarily controlled by gonadotropins with contributions by various growth factors. Gonadotropins through their receptors switch on cyclic AMP signaling pathways that recruit NR5A, GATA and often CREB, NR4A, or Sp1 transcription factors to regulatory regions of each gene's promoter to elicit transcription. The specific combination of transcription factors involved depends on the cellular context. In this review, we summarize current understanding of the factors that control transactivation of the STARD1, CYP11A1 and gonadal HSD3B genes in Leydig cells in the testis and the theca, differentiating granulosa and luteal cells in the ovary.

Gonadal transactivation of STARD1, CYP11A1 and HSD3B.

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

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Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism.

The ovulatory LH surge induces rapid up-regulation of Cyp11a1 in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic controls including histone modifications and DNA methylation in the promoter region are associated with the rapid increase of Cyp11a1 gene expression after LH surge. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and 4 h and 12 h after human (h)CG injection. Cyp11a1 mRNA levels rapidly increased after hCG injection, reached a peak at 4 hours, and then remained elevated until 12 hours. DNA methylation status in the Cyp11a1 proximal promoter region was hypomethylated and did not change at any of the observed times after hCG injection. Chromatin immunoprecipitation assays revealed that the levels of trimethylation of lysine 4 on histone H3 (H3K4me3), an active mark for transcription, increased, whereas the levels of H3K9me3 and H3K27me3, which are marks associated with repression of transcription, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin immunoprecipitation assays also showed that the binding activity of CAATT/enhancer-binding protein-β to the Cyp11a1 proximal promoter increased after hCG injection. Luciferase assays revealed that the CAATT/enhancer-binding protein-β-binding site had transcriptional activity and contributed to basal and cAMP-induced Cyp11a1 expression. These results suggest that changes in histone modification and chromatin structure in the Cyp11a1 proximal promoter are involved in the rapid increase of Cyp11a1 gene expression in GCs undergoing luteinization during ovulation.

Epigenetic Changes of the Cyp11a1 Promoter Region in Granulosa Cells Undergoing Luteinization During Ovulation in Female Rats.

The first step of steroid biosynthesis is catalyzed by cytochrome P450scc, encoded by CYP11A1. To achieve steroidogenic tissue-specific inactivation of genes in vivo by the Cre-loxP approach, we used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the tissues that produce steroids. The resulting SCC-Cre mice express high levels of Cre in the adrenal cortex and gonads at the same sites as that for the endogenous CYP11A1 expression. In addition, Cre activity was found in the diencephalon and midbrain. In the developing brain, the Cre activity was first detected in the embryonic day 10.5. Our study is the first to show that the 4.4-kb CYP11A1 promoter is transcriptionally active in the brain in vivo. genesis 45:59–65, 2007. © 2007 Wiley-Liss, Inc.

Human CYP11A1 promoter drives Cre recombinase expression in the brain in addition to adrenals and gonads.

CYP11A1 encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression of CYP11A1 in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controlling CYP11A1 expression is steroidogenic factor-1 (SF-1), but the transcriptional regulation of CYP11A1 in the brain is unclear. We recently used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional-specific expression of Cre reporter in the SCC-Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF-1 binding site did not affect brain CYP11A1 promoter activity. The upstream SF-1 binding site, however, is required for CYP11A1 promoter function in the embryonic adrenals. The 3.8-kb promoter, like the 4.4-kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7-kb promoter was only observed in the caudal part of midbrain. This suggests that the 5'-flanking region between 3.8 and 2.7 kb contains a crucial element for activation of CYP11A1 promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that control CYP11A1 expression in the brain and embryonic adrenals.

Hui-Ting Lin

Papers

Human CYP11A1 promoter drives Cre recombinase expression in the brain in addition to adrenals and gonads.

Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals.