Synthia H. Mellon

Neurosteroids: Biosynthesis and Function of These Novel Neuromodulators☆

Over the past decade, it has become clear that the brain is a steroidogenic organ. The steroids synthesized by the brain and nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions, not through classic steroid hormone nuclear receptors, but through ion-gated neurotransmitter receptors. This paper summarizes what is known about the biosynthesis of neurosteroids, the enzymes mediating these reactions, their localization during development and in the adult, and their function and mechanisms of action in the developing and adult central and peripheral nervous systems. The expression of the steroidogenic enzymes is developmentally regulated, with some enzymes being expressed only during development, while others are expressed during development and in the adult. These enzymes are expressed in both neurons and glia, suggesting that these two cell types must work in concert to produce the appropriate active neurosteroid. The functions attributed to specific neurosteroids include modulation of GABA(A) and NMDA function, modulation of sigma receptor function, regulation of myelinization, neuroprotection, and growth of axons and dendrites. Neurosteroids have also been shown to modulate expression of particular subunits of GABA(A) and NMDA receptors, providing additional sites at which these compounds can regulate neural function. The pharmacological properties of specific neurosteroids are described, and potential uses of neurosteroids in specific neuropathologies and during normal aging in humans are also discussed.

Neurobiological and Neuropsychiatric Effects of Dehydroepiandrosterone (DHEA) and DHEA Sulfate (DHEAS)

DHEA and DHEAS are steroids synthesized in human adrenals, but their function is unclear. In addition to adrenal synthesis, evidence also indicates that DHEA and DHEAS are synthesized in the brain, further suggesting a role of these hormones in brain function and development. Despite intensifying research into the biology of DHEA and DHEAS, many questions concerning their mechanisms of action and their potential involvement in neuropsychiatric illnesses remain unanswered. We review and distill the preclinical and clinical data on DHEA and DHEAS, focusing on (i) biological actions and putative mechanisms of action, (ii) differences in endogenous circulating concentrations in normal subjects and patients with neuropsychiatric diseases, and (iii) the therapeutic potential of DHEA in treating these conditions. Biological actions of DHEA and DHEAS include neuroprotection, neurite growth, and antagonistic effects on oxidants and glucocorticoids. Accumulating data suggest abnormal DHEA and/or DHEAS concentrations in several neuropsychiatric conditions. The evidence that DHEA and DHEAS may be fruitful targets for pharmacotherapy in some conditions is reviewed.

Neurosteroid biosynthesis: genes for adrenal steroidogenic enzymes are expressed in the brain.

To determine if neurosteroids (steroids synthesized in the brain) are produced by enzymes found in steroidogenic tissues, we determined if mRNA for five steroidogenic enzymes could be detected in brain tissues or cultured cells. We detected mRNAs for adrenodoxin, P450scc (cholesterol side-chain cleavage enzyme) and P450c11 beta (11 beta-hydroxylase) but not for P450c17 (17 alpha-hydroxylase/17,20 lyase) or P450c11AS (aldosterone synthase) in rat brains and cultures of rat glial cells. P450scc mRNA abundance in brain or primary glial cultures was approximately 0.01% of that found in the adrenal, but more P450scc mRNA was detected in C6 glial cells. Both P450scc and P450c11 beta mRNAs were most abundant in the cortex, but there were region-specific differences for both mRNAs, and sex-specific differences for P450c11 beta mRNA. P450scc mRNA was equally abundant in mixed glial cultures containing both astrocytes and oligodendrocytes as in astrocyte-enriched cultures, and P450scc immunoreactivity co-localized with GFAP immunoreactivity in cultured astrocytes. P450c11 beta mRNA was not detected in the mixed primary glial cultures for the C6 glioma cell line that synthesize P450scc mRNA, suggesting that glial cells do not synthesize P450c11 beta mRNA. Thus some of the same enzymes involved in steroidogenesis in classic endocrine tissues are found in a cell-specific and region-specific fashion in the brain. Neurosteroids may be derivatives of known classic steroids, and/or may function through non-classic steroid hormone receptors, such as GABAA, N-methyl-D-aspartate, and corticosterone receptors.

Niemann–Pick type C disease involves disrupted neurosteroidogenesis and responds to allopregnanolone

Niemann–Pick type C (NP-C) disease is a fatal, autosomal recessive, childhood neurodegenerative disease. The NP-C mouse recapitulates the cholesterol and sphingolipid storage, onset of neurological deficits, histopathological lesions, Purkinje cell loss and early death typical of the most severe form of human NP-C. Neurosteroids, steroids made in the brain, affect neuronal growth and differentiation, and modulate neurotransmitter receptors. Disordered cholesterol trafficking might disrupt neurosteroidogenesis, thereby contributing to the NP-C phenotype. Here we show that NP-C mouse brain contains substantially less neurosteroid than wild-type brain and has an age-related decrease in the ability to synthesize 5α-dihydroprogesterone and allopregnanolone. Immunohistochemical assessment confirms a decrease in expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase, especially in cerebellum. Neonatal administration of allopregnanolone delays the onset of neurological symptoms, increases Purkinje and granule cell survival, reduces cortical GM2 and GM3 ganglioside accumulation and doubles the lifespan of NP-C mice. Earlier administration increases effectiveness of treatment. Decreased production of allopregnanolone apparently contributes to the pathology of NP-C; thus, neurosteroid treatment may be useful in ameliorating progression of the disease.

Biosynthesis and action of neurosteroids

Over the past decade, it has become clear that the brain, like the gonad, adrenal and placenta, is a steroidogenic organ. However, unlike classic steroidogenic tissues, the synthesis of steroids in the nervous system requires the coordinate expression and regulation of the genes encoding the steroidogenic enzymes in several different cell types (neurons and glia) at different locations in the nervous system, and at distances from the cell bodies. The steroids synthesized by the brain and nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions, not through classic steroid hormone nuclear receptors, but through other mechanisms such as through ion gated neurotransmitter receptors, or through direct or indirect modulation of other neurotransmitter receptors. We have briefly summarized the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their localization during development and in the adult, and the regulation of their expression, highlighting both similarities and differences between expression in the brain and in classic steroidogenic tissues.

Analyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance

Telomeres are the DNA-protein complexes that protect the ends of eukaryotic chromosomes. The cellular enzyme telomerase counteracts telomere shortening by adding telomeric DNA. A growing body of literature links shorter telomere length and lower telomerase activity with various age-related diseases and earlier mortality. Thus, leukocyte telomere length (LTL) and telomerase activity are emerging both as biomarkers and contributing factors for age-related diseases. However, no clinical study has directly examined telomerase activity and telomere length in different lymphocyte subtypes isolated from the same donors, which could offer insight into the summary measure of leukocyte telomere maintenance. We report the first quantitative data in humans examining both levels of telomerase activity and telomere length in four lymphocyte subpopulations from the same donors-CD4+, CD8+CD28+ and CD8+CD28- T cells and B cells, as well as total PBMCs-in a cohort of healthy women. We found that B cells had the highest telomerase activity and longest telomere length; CD4+ T cells had slightly higher telomerase activity than CD8+CD28+ T cells, and similar telomere length. Consistent with earlier reports that CD8+CD28- T cells are replicatively senescent cells, they had the lowest telomerase activity and shortest telomere length. In addition, a higher percentage of CD8+CD28- T cells correlated with shorter total PBMC TL (r=-0.26, p=0.05). Interestingly, telomerase activities of CD4+ and CD8+CD28+ T cells from the same individual were strongly correlated (r=0.55, r<0.001), indicating possible common mechanisms for telomerase activity regulation in these two cell subtypes. These data will facilitate the understanding of leukocyte aging and its relationship to human health.

Molecular Mechanism of Suppression of Testicular Steroidogenesis by Proinflammatory Cytokine Tumor Necrosis Factor Alpha

ABSTRACT Tumor necrosis factor alpha (TNF-α) has been demonstrated to inhibit steroidogenesis in Leydig cells at the transcriptional level of steroidogenic enzymes. However, the molecular mechanism of this observed gene repression is not well understood. We now demonstrate that nuclear factor κB (NF-κB) activated by TNF-α inhibits the transactivation of orphan nuclear receptors, which regulate the expression of steroidogenic-enzyme genes. TNF-α treatment suppressed the luteinizing-hormone-induced or Nur77/SF-1-stimulated promoter activity of steroidogenic-enzyme genes in Leydig cells. The TNF-α-mediated gene suppression was blocked by treatment with an inhibitor of NF-κB. In addition, overexpression of the p65 (RelA) subunit of NF-κB showed the same effect as TNF-α and inhibited Nur77 transactivation, suggesting the involvement of NF-κB activation in the observed gene repression. Physical association of Nur77 with p65 was revealed by mammalian two-hybrid, GST pull-down, and coimmunoprecipitation analyses. The NF-κB inhibition of Nur77 transactivation was likely due to the competition of p65 for Nur77 binding with coactivators. Finally, chromatin immunoprecipitation assays revealed that TNF-α treatment caused the recruitment of NF-κB to the promoter of the steroidogenic-enzyme P450c17 gene, supporting the hypothesis that the TNF-α-mediated gene repression involves NF-κB inhibition of the transcriptional activity of Nur77 and other orphan nuclear receptors. These findings provide a molecular mechanism underlying the inhibition of testicular steroidogenesis by proinflammatory cytokines.

Biosynthesis of neurosteroids and regulation of their sysnthesis

The brain, like the gonads, adrenal glands, and placenta, is a steroidogenic organ. The steroids synthesized by the brain and by the nervous system, given the name neurosteroids, have a wide variety of diverse functions. In general, they mediate their actions not through classic steroid hormone nuclear receptors but through ion-gated neurotransmitter receptors. This chapter summarizes the biochemistry of the enzymes involved in the biosynthesis of neurosteroids, their localization during development and in adulthood, and the regulation of their expression, highlighting both similarities and differences between expression in the brain and in classic steroidogenic tissues.

A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.

Protein Phosphatase 2A and Phosphoprotein SET Regulate Androgen Production by P450c17

Cytochrome P450c17 catalyzes 17α-hydroxylation needed for cortisol synthesis and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of P450c17 specifically increases 17,20 lyase activity, but the physiological factors regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with the phosphatase inhibitors okadaic acid, fostriecin, and cantharidin increased 17,20 lyase activity, suggesting involvement of protein phosphatase 2A (PP2A) or 4 (PP4). PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells, but neither affected 17α-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was concentration-dependent, could be inhibited by okadaic acid, and was restored by endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17, and suppression of PP2A by small interfering RNA increased 17,20 lyase activity. Phosphoprotein SET found in adrenals inhibited PP2A, but not PP4, and fostered 17,20 lyase activity. The identification of PP2A and SET as post-translational regulators of androgen biosynthesis suggests potential additional mechanisms contributing to adrenarche and hyperandrogenic disorders such as polycystic ovary syndrome.