Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.     

Epipregnanolone Acts as a Partial Agonist on a Common Neurosteroid Modulatory Site of the GABAA Receptor Complex in Avian CNS

Neurosteroid modulatory sites present in the GABA_A receptor complex in chick optic lobe were investigated, in order to evaluate whether allopregnanolone and alphaxalone act through a common site of action. Results showed that either allopregnanolone or alphaxalone present a single-component enhancement of [³H]flunitrazepam binding with EC₅₀ of 1.18 ± 0.12 and 6.56 ± 0.86 M and E_max of 82.18 ± 5.80 and 62.98 ± 3.73 %, respectively. Epipregnanolone behaved as a partial agonist of these steroid modulatory sites with EC₅₀ of 0.49 ± 0.15 M and E_max 12.34 ± 1.03%. Moreover, the addition of 16 M epipregnanolone to either allopregnanolone or alphaxalone decreased EC₅₀ values to 0.54 ± 0,09 and 1.24 ± 0.25 M respectively, while E_max values were not significantly affected. On the other hand, additivity experiments disclosed that a maximal concentration (16 M) of alphaxalone in the presence of allopregnanolone failed to enhance [³H]flunitrazepam binding in excess of that produced by allopregnanolone alone. Results indicate that not only allopregnanolone and alphaxalone act through a common site of action, but such site is highly stereospeciflc with regard to the neurosteroid spatial configuration.     

Defective cholesterol trafficking in Niemann-Pick C-deficient cells

Pathways of intracellular cholesterol trafficking are poorly understood at the molecular level. Mutations in Niemann-Pick C (NPC) proteins, NPC1 and NPC2, however, have led to insights into the mechanism by which endocytosed cholesterol is exported from late endosomes/lysosomes (LE/L). Mutations in NPC1, a multi-spanning membrane protein of LE/L, or mutations in NPC2, a soluble luminal protein of LE/L, cause the neurodegenerative disorder NPC disease. This review focuses on data supporting a model in which movement of cholesterol out of LE/L is mediated by the sequential action of the two NPC proteins. We also discuss potential therapies for NPC disease, including evidence that treatment of NPC-deficient mice with the cholesterol-binding compound, cyclodextrin, markedly attenuates neurodegeneration, and increases life-span, of NPC1-deficient mice.     

Allopregnanolone levels are reduced in temporal cortex in patients with Alzheimer's disease compared to cognitively intact control subjects

The neurosteroid allopregnanolone has pronounced neuroprotective actions, increases myelination, and enhances neurogenesis. Evidence suggests that allopregnanolone dysregulation may play a role in the pathophysiology of Alzheimer's disease (AD) and other neurodegenerative disorders. Our prior data demonstrate that allopregnanolone is reduced in prefrontal cortex in male patients with AD compared to male cognitively intact control subjects, and inversely correlated with neuropathological disease stage (Braak and Braak). We therefore determined if allopregnanolone levels are also reduced in AD patients compared to control subjects in temporal cortex, utilizing a larger set of samples from both male and female patients. In addition, we investigated if neurosteroids are altered in subjects who are APOE4 allele carriers. Allopregnanolone, dehydroepiandrosterone (DHEA), and pregnenolone levels were determined in temporal cortex postmortem samples by gas chromatography/mass spectrometry, preceded by high performance liquid chromatography (40 subjects with AD/41 cognitively intact control subjects). Allopregnanolone levels are reduced in temporal cortex in patients with AD (median 2.68 ng/g, n = 40) compared to control subjects (median 5.64 ng/g, n = 41), Mann–Whitney p = 0.0002, and inversely correlated with Braak and Braak neuropathological disease stage (Spearman r = − 0.38, p = 0.0004). DHEA and pregnenolone are increased in patients with AD compared to control subjects. Patients carrying an APOE4 allele demonstrate reduced allopregnanolone levels in temporal cortex (Mann–Whitney p = 0.04). In summary, our findings indicate that neurosteroids are altered in temporal cortex in patients with AD and related to neuropathological disease stage. In addition, the APOE4 allele is associated with reduced allopregnanolone levels. Neurosteroids may be relevant to the neurobiology and therapeutics of AD.     

Non-stereo-selective cytosolic human brain tissue 3-ketosteroid reductase is refractory to inhibition by AKR1C inhibitors

Cerebral 3α-hydroxysteroid dehydrogenase (3α-HSD) activity was suggested to be responsible for the local directed formation of neuroactive 5α,3α-tetrahydrosteroids (5α,3α-THSs) from 5α-dihydrosteroids. We show for the first time that within human brain tissue 5α-dihydroprogesterone and 5α-dihydrotestosterone are converted via non-stereo-selective 3-ketosteroid reductase activity to produce the respective 5α,3α-THSs and 5α,3β-THSs. Apart from this, we prove that within the human temporal lobe and limbic system cytochrome P450c17 and 3β-HSD/Δ^5–4 ketosteroid isomerase are not expressed. Thus, it appears that these brain regions are unable to conduct de novo biosynthesis of Δ⁴-3-ketosteroids from Δ⁵-3β-hydroxysteroids. Consequently, the local formation of THSs will depend on the uptake of circulating Δ⁴-3-ketosteroids such as progesterone and testosterone. 3α- and 3β-HSD activity were (i) equally enriched in the cytosol, (ii) showed equal distribution between cerebral neocortex and subcortical white matter without sex- or age-dependency, (iii) demonstrated a strong and significant positive correlation when comparing 46 different specimens and (iv) exhibited similar sensitivities to different inhibitors of enzyme activity. These findings led to the assumption that cerebral 3-ketosteroid reductase activity might be catalyzed by a single enzyme and is possibly attributed to the expression of a soluble AKR1C aldo-keto reductase. AKR1Cs are known to act as non-stereo-selective 3-ketosteroid reductases; low AKR1C mRNA expression was detected. However, the cerebral 3-ketosteroid reductase was clearly refractory to inhibition by AKR1C inhibitors indicating the expression of a currently unidentified enzyme. Its lack of stereo-selectivity is of physiological significance, since only 5α,3α-THSs enhance the effect of GABA on the GABA_A receptor, whereas 5α,3β-THSs are antagonists.     

Allopregnanolone increase in striatal N-methyl-D-aspartic acid evoked [3H]dopamine release is estrogen and progesterone dependent

1. The neurosteroids are compounds derived from steroid hormones and synthesized in the nervous system. They can modulate different neurotransmitter pathways. In previous work we demonstrated that progesterone modulates dopamine release induced by the glutamatergic agonist N-methyl-D-aspartic acid (NMDA).2. The aim of this work was to evaluate a possible modulatory role of the progesterone metabolite allopregnanolone on NMDA-evoked [³H]dopamine release from corpus striatum slices obtained from cycling and ovariectomized female rats.3. We used a dynamic superfusion method to evaluate the release of [³H]dopamine. Allopregnanolone at 50–600 nM was added to the superfusion buffer (Krebs–Ringer–bicarbonate–glucose, pH 7.4, with constant O₂/CO₂ gassing). The results are expressed as a percentage over basal [³H]dopamine loaded by the tissue.4. Allopregnanolone (50 and 100 nM) increased the NMDA-evoked[³H]dopamine release from estrus rats. The remaining doses did not show significant changes in the pattern of release. This effect was not observed in diestrus rats. The ovariectomy abolished the facilitatory effect of allopregnanolone on NMDA-evoked 2 [³H]dopamine release.5. Subcutaneous administration of exogenous estrogen (25 mg/rat) and progesterone (1 mg/rat) restored the facilitatory effect on dopaminergic input.6. These results suggest that allopregnanolone is a neurosteroid able to modulate dopamine release in an ovarian-hormone-fluctuation-dependent manner and provide further support for a role of allopregnanolone as a modulator of glutamatergic–dopaminergic interaction in the corpus striatum.     

Sensitivity to the locomotor-stimulant effects of ethanol and allopregnanolone: a quantitative trait locus study of common genetic influence

Previous studies have suggested that common genetic mechanisms influence sensitivity to the locomotor-stimulant effects of ethanol and allopregnanolone. We conducted two quantitative trait locus (QTL) studies to identify chromosomal regions that harbor genes that influence locomotor response to ethanol (2 g/kg) and allopregnanolone (17 mg/kg) using F2 crosses between C57BL/6J and DBA/2J mice. Because our previous data from the BXD recombinant inbred strains had indicated that chromosome 2 contained QTL for sensitivity to the locomotor-stimulant effects of both ethanol and allopregnanolone, we also tested reciprocal chromosome 2 congenic strains for sensitivity to the locomotor-stimulant effects of both drugs. The F2 analysis for ethanol sensitivity identified significant QTL on chromosomes 1 and 2 and suggestive QTL on chromosomes 5 and 9. The analysis of the allopregnanolone F2 study identified suggestive QTL on chromosomes 3, 5 and 12. Suggestive evidence for a female-specific QTL on chromosome 2 was also found. The studies of congenic mouse strains indicated that both the congenic strains captured one or more QTL for sensitivity to the locomotor-stimulant effects of both ethanol (2 g/kg) and allopregnanolone (17 mg/kg). When Fisher's method was used to combine the P values for the RI, F2 and congenic studies of the chromosome 2 QTL, cumulative probability scores of 9.6 x 10(-15) for ethanol and 7.7 x 10(-7) for allopregnanolone were obtained. These results confirm the presence of QTL for ethanol and allopregnanolone sensitivity in a common region of chromosome 2 and suggest possible pleiotropic genetic influence on sensitivity to these drugs.     

神经活性甾体别孕烯醇酮对脑皮质神经元损伤的保护作用

目的：利用N-甲基-D-天门冬氨酸（NMDA）诱发新生小鼠脑皮质神经元损伤模型,探讨神经活性甾体别孕烯醇酮对脑皮质神经元的保护作用及其机制。方法：应用RT-PCR和Western blot法检测别孕烯醇酮对β2-γ-氨基丁酸受体（β2-GABA-R）表达和对蛋白激酶B（PKB,又称为Akt）磷酸化的影响。应用Western blot和DNA-Ladder方法检测NMDA诱发的神经元凋亡及别孕烯醇酮对NMDA诱发凋亡的影响。结果：Western blot和RT-PCR分析表明0.5×10-6mol/L-5×10-6mol/L别孕烯醇酮使Akt磷酸化增加并促进β2-GABA-R mRNA的表达。1×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元有抗凋亡作用,但5×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元使NMDA诱发的DNA-Ladder减弱明显,并能有效抵抗NMDA诱发的活化型PRAP、Caspase-3、Caspase-9的增加。结论：别孕烯醇酮可通过促进β2-GABA-R表达和增加Akt磷酸化抵抗NMDA诱发的脑皮质神经元凋亡。     

Neurosteroid modulation of N-methyl-D-aspartate receptors: molecular mechanism and behavioral effects

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-d-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.