Neurosteroid Synthesis by Cytochrome P450-Containing Systems Localized in the Rat Brain Hippocampal Neurons: N-Methyl-d-Aspartate and Calcium-Dependent Synthesis

Abstract

Neurosteroidogenesis has not been well elucidated due to the very low level of steroidogenic proteins in the brain. Here we report the first demonstration of the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in the adult rat hippocampus. Significant localization of cytochrome P450scc was observed in pyramidal neurons and granule neurons by means of immunohistochemical staining of slices. We also observed the colocalization, in hippocampal neurons, of P450scc with redox partners, hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein. The distributions of astroglial cells and oligodendroglial cells showed very different patterns from that of the P450scc-containing cells. The expression of P450scc, redox partners, the sulfotransferase, and steroidogenic acute regulatory protein was also confirmed by Western blot analysis. The process of active neurosteroidogenesis was stimulated by exposing neurons to N-methyl-D-aspartate. Upon stimulation with N-methyl-D-aspartate, Ca(2+) influx through the N-methyl-D-aspartate subtype of glutamate receptors occurred, and significant net production of pregnenolone and pregnenolone sulfate was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of N-methyl-D-aspartate receptors, by Ca(2+) depletion, or by the addition of an inhibitor of P450scc. Upon stimulation with N-methyl-D-aspartate, the processing of full-length steroidogenic acute regulatory protein (37-kDa) to the truncated 30-kDa steroidogenic acute regulatory protein was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication.

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017α and P450 aromatase localized in neurons

“…Stimulation of net estradiol production with NMDA was completely suppressed by the application of 50 M MK-801 (a specific blocker of NMDA receptors, Sigma). This enhancement in estradiol synthesis may be due to an increase in the NMDA receptor-mediated Ca 2ϩ influx that drives the transport of cholesterol to the inner membrane of mitochondria, followed by a cascade of steroidogenesis (7). The basal concentration of DHEA was also measured in the hippocampus and plasma.…”

“…In our previous work, the steroid synthesis was triggered by exposing neurons to NMDA, which induced a Ca 2ϩ influx through the NMDA receptors and resulted in the significant production of PREG(s) (5,7). A pool of full-length (37-kDa) steroidogenic acute regulatory protein (StAR) was processed to the truncated 30-kDa StAR upon NMDA stimulation (7).…”

“…In our previous work, the steroid synthesis was triggered by exposing neurons to NMDA, which induced a Ca 2ϩ influx through the NMDA receptors and resulted in the significant production of PREG(s) (5,7). A pool of full-length (37-kDa) steroidogenic acute regulatory protein (StAR) was processed to the truncated 30-kDa StAR upon NMDA stimulation (7). The expression of essential steroidogenic proteins [StAR, P450scc (CYP11A1), and 3␤-HSD] in the hippocampal principal neurons was demonstrated by means of immunostaining and Western blot analysis (7)(8)(9) or by in situ hybridization (17,22).…”

“…Evidence is emerging that estrogen exerts not only the chronic/genomic effects but also a rapid/nongenomic influence on hippocampal synaptic plasticity (2,5). In addition to endocrine-derived hormones, recent experiments have demonstrated that hippocampal neurons may also be exposed to locally synthesized brain neurosteroids, such as pregnenolone (PREG) and its sulfated ester (6)(7)(8)(9). Dehydroepiandrosterone (DHEA) has been found in the mammalian brain at concentrations greater than that in plasma (6,10).…”

Acute inhibition of estradiol synthesis impacts vestibulo-ocular reflex adaptation and cerebellar long-term potentiation in male rats

“…In addition to gonadal origin, E2 is locally synthesized in the central nervous system from cholesterol (Baulieu 1997;Compagnone and Mellon 2000), by an aromatasedependent conversion of testosterone (Kimoto et al 2001). Local E2 is a region-specific neurosteroid and can play an important role in modulating neuronal activity due to its ability to rapidly reach higher concentrations than the gonadal hormone (Hojo et al 2009;Mukai et al 2010).…”