Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3alpha,5alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3alpha,5alpha-tetrahydroprogesterone (allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5alpha-dihydroprogesterone. Furthermore, the presence of 3beta-, 11beta-, 17alpha-, and 20alpha-hydroxylated metabolites of 3alpha,5alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3alpha,5alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids.

Mis-processing of amyloid precursor protein (APP) and aberrant phosphorylation of the microtubule-associated protein tau are key pathogenic mechanisms in Alzheimer's disease (AD). The metabolism of APP and phosphorylation of tau, both highly regulated processes, are intrinsically linked to neuronal function and survival. While full-length APP is implicated in normal cell physiology (Reinhard et al. 2005), its sequential proteolysis by a, b and c-secretases results in the generation of either neuroprotective or neurotoxic peptides. Cleavage by a-secretase yields nonamyloidogenic secreted aAPP (saAPP) and an 83-residue C-terminal fragment (C83); alternative cleavage by b-secretase (BACE) generates secreted bAPP (sbAPP) and a 99-residue product (C99); the latter can be further catabolized by c-secretase in the so-called 'amyloidogenic pathway' to generate amyloid b (Ab). While aggregates of Ab give rise to the characteristic senile plaques found in AD-affected brains, both C99 and soluble Ab have intrinsic neurotoxic properties (Roselli et al. 2005;Yankner et al., 1989 Abbreviations used: AD, Alzheimer's disease; APP, amyloid precursor protein; Ab, amyloid beta; C99, 99-residue C-terminal fragment of APP; cdk5, cyclin-dependent kinase 5; DIV, days in vitro; EGFP, enhanced green fluorescent protein; GC, glucocorticoid(s); GR, glucocorticoid receptor; GSK3, glycogen synthase kinase 3; htau, human tau; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; Nct, nicastrin; PBS, phosphate-buffered saline; PS1, presenilin 1. AbstractAmyloid precursor protein (APP) mis-processing and aberrant tau hyperphosphorylation are causally related to the pathogenesis and neurodegenerative processes that characterize Alzheimer's disease (AD). Abnormal APP metabolism leads to the generation of neurotoxic amyloid beta (Ab), whereas tau hyperphosphorylation culminates in cytoskeletal disturbances, neuronal dysfunction and death. Many AD patients hypersecrete glucocorticoids (GC) while neuronal structure, function and survival are adversely influenced by elevated GC levels. We report here that a rat neuronal cell line (PC12) engineered to express the human ortholog of the tau protein (PC12-htau) becomes more vulnerable to the toxic effects of either Ab or GC treatment. Importantly, APP metabolism in GC-treated PC12-htau cells is selectively shifted towards increased production of the pro-amyloidogenic peptide C99. Further, GC treatment results in hyperphosphorylation of human tau at AD-relevant sites, through the cyclin-dependent kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein kinases. Pulse-chase experiments revealed that GC treatment increased the stability of tau protein rather than its de novo synthesis. GC treatment also induced accumulation of transiently expressed EGFP-tau in the neuronal perikarya. Together with previous evidence showing that Ab can activate cyclin-dependent kinase 5 and GSK3, these results uncover a potential mechanism through which GC may contribute to AD neuropathology.

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Withdrawal from long-term dosing with exogenous progesterone precipitates increased anxiety-linked changes in behavior in animal models due to the abrupt decrease in brain concentration of allopregnanolone (ALLO), a neuroactive metabolite of progesterone. We show that a withdrawal-like effect also occurs during the late diestrus phase (LD) of the natural ovarian cycle in rats, when plasma progesterone and ALLO are declining but estrogen secretion maintains a stable low level. This effect at LD was prevented by short-term treatment with low dose fluoxetine. During LD, but not at other stages of the estrous cycle, exposure to anxiogenic stress induced by whole body vibration at 4 Hz for 5 min evoked a significant decrease in tail flick latency (stress-induced hyperalgesia) and a decrease in the number of Fos-positive neurons present in the periaqueductal gray (PAG). The threshold to evoke fear-like behaviors in response to electrical stimulation of the dorsal PAG was lower in the LD phase, indicating an increase in the intrinsic excitability of the PAG circuitry. All these effects were blocked by short-term administration of fluoxetine (2 × 1.75 mg kg(-1) i.p.) during LD. This dosage increased the whole brain concentration of ALLO, as determined using gas chromatography-mass spectrometry, but was without effect on the extracellular concentration of 5-HT in the dorsal PAG, as measured by microdialysis. We suggest that fluoxetine-induced rise in brain ALLO concentration during LD offsets the sharp physiological decline, thus removing the trigger for the development of anxiogenic withdrawal effects.

Little is known about the origin of the neuroactive steroids dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PregS) in the brain or of their subsequent metabolism. Using rat brain perfusion in situ, we have found 3 H-PregS to enter more rapidly than 3 H-DHEAS and both to undergo extensive (> 50%) desulphation within 0.5 min of uptake. Enzyme activity for the steroid sulphatase catalysing this deconjugation was enriched in the capillary fraction of the blood-brain barrier and its mRNA expressed in cultures of rat brain endothelial cells and astrocytes. Although permeability measurements suggested a net efflux, addition of the efflux inhibitors GF120918 and/or MK571 to the perfusate reduced rather than enhanced the uptake of 3 H-DHEAS and 3 H-PregS; a further reduction was seen upon the addition of unlabelled steroid sulphate, suggesting a saturable uptake transporter.Analysis of brain fractions after 0.5 min perfusion with the 3 Hsteroid sulphates showed no further metabolism of PregS beyond the liberation of free steroid pregnenolone. By contrast, DHEAS underwent 17-hydroxylation to form androstenediol in both the steroid sulphate and the free steroid fractions, with some additional formation of androstenedione in the latter. Our results indicate a gain of free steroid from circulating steroid sulphates as hormone precursors at the blood-brain barrier, with implications for ageing, neurogenesis, neuronal survival, learning and memory.