Abstract
There is emerging evidence from animal studies for a neuroprotective role of sex steroids in neurodegenerative diseases, but studies in human brain are lacking. We have carried out an extensive study of the neurosteroid biosynthetic pathways in substantia nigra (SN), caudate nucleus (CN) and putamen (PU) of 7 Parkinson's disease (PD) patients and 7 matched controls. The mRNA levels of 37 genes including neurosteroid biosynthetic enzymes, hormone receptors and the neurosteroid‐modulated γ‐amino‐butyric acid ‐A (GABA‐A) receptor subunits were analyzed by quantitative PCR (qPCR). In the SN, we found downregulation of 5α‐reductase type 1 (5α‐R1), sulfotransferase 2B1 (SULT2B1) and some GABA‐A receptor subunits (α4, β1) while in the CN, upregulation of 3α‐hydroxysteroid dehydrogenase type 3 (3α‐HSD3) and α4 GABA‐A receptor subunit (22‐fold) was observed. No significant differences were found in the PU. These data imply an involvement of pregnane steroids and changes in GABAergic neurotransmission in the neurodegenerative process and suggest that neurosteroids may deserve further investigation as potential therapeutic agents in PD.
Keywords: 3αlpha‐hydroxysteroid dehydrogenase, 5αlphareductase, GABA‐A receptors, gene expression, neurosteroids, Parkinson's disease
INTRODUCTION
Sex steroids such as estrogens, androgens and progesterone that are synthesized de novo in the brain are called neurosteroids (1). The enzymes responsible for the synthesis of neurosteroids are widely expressed in the central nervous system (CNS) 22, 43, 57.
Neurosteroids have a variety of functions including neuroprotection and neurotrophic actions (15). The neuroprotective effects are mediated by the interaction of neurosteroids with their nuclear receptors, which can lead, for example, to increased expression of neurotrophic factors such as brain‐derived neurotrophic factor and insulin‐like growth factor 1 26, 46. Some neurosteroids have also shown neurotransmitter modulatory action in the CNS and they are referred to as “neuroactive.” For example, allopregnanolone, a metabolite of progesterone, is considered the most potent allosteric modulator of γ‐amino‐butyric acid ‐A (GABA‐A) receptors (42).
Many animal models for Parkinson's disease (PD) have suggested a role for sex steroids as neuroprotective agents for dopaminergic cells 7, 29. In mice treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) or methamphetamine (MA), estrogens were found to protect dopaminergic neurons from degeneration 10, 18, 51. Evidence for a protective effect of progesterone on these neurons has also been found in MPTP but not in MA treated mice 11, 30, 45.
In studies of human brain, Bixo et al (4) found relatively high levels of progesterone and its metabolites 5α‐dehydro progesterone (5α‐DHP) and allopregnanolone in the substantia nigra (SN) and caudate nucleus (CN) of female control subjects, compared with other brain areas, indicating that synthesis and accumulation of these compounds take place in the nigrostriatal dopaminergic system.
Previously, we found reduced levels of allopregnanolone and 5α‐DHP in the cerebrospinal fluid (CSF) of PD patients suggesting a role for these progesterone metabolites in the disease (19). Further investigation of the mRNA expression of 5α‐reductase type 1 (5α‐R1) and 3α‐hydroxysteroid dehydrogenase type 2 (3α‐HSD2), the enzymes which synthesize allopregnanolone revealed that 5α‐R1 was significantly reduced in peripheral blood mononuclear cells (PMBC) of PD patients, suggesting a defect in the enzymatic machinery that regulates the metabolism of progesterone (40).
These results warrant further investigation of the biosynthesis and metabolism pathways of steroids in the CNS. Identifying potential alterations in neurosteroid biosynthesis and metabolism in brain areas affected by PD may be beneficial to understand the role of neurosteroids in this disease and identify potential targets for new therapeutic approaches.
In this study, the gene expression of the enzymes involved in the synthesis and metabolism of neurosteroids and their receptors in PD was studied, together with the GABA‐A receptors subunits on which they exert their modulation in the brain.
We present data on the expression of a total of 37 genes analyzed by quantitative PCR (qPCR). Immunohistochemistry (IHC) for 5α‐R was also carried out to localize the changes in expression of this enzyme.
MATERIALS AND METHODS
Subjects
PD patients (n = 7) were clinically and neuropathologically diagnosed. All of them received DA replacement therapies during the course of their disease. Subjects without neurological or psychiatric disorders and without neuropathological alterations (n = 7) were included as controls. The controls did not exceed a Braak stage pathology score for neurofibrillary tangles of 2 (8). Postmortem human brain tissue was obtained from the Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam (NBB). Written informed consent for a brain autopsy and the use of the material and clinical information for research purposes was acquired by NBB. Clinicopathological information is summarized in Table S1.
Subjects were chosen to be matched for age, sex, postmortem interval and brain pH as closely as possible. As the pH of the CSF is a good predictor for RNA integrity (59), samples with a CSF pH below 6.3 were excluded.
In addition, we included only patients who were not treated with corticosteroids, indometacin, benzodiazepine and hormonal therapies in the last 3 months before death. Only subjects from whom freshly frozen tissue samples of the CN, SN and putamen (PU) were available were included. No significant differences between PD and control groups for age, pH, RNA integrity, postmortem interval or brain weight were found (Mann–Whitney U‐test, Table S1).
RNA isolation, reverse transcription and qPCR
SN was reproducibly dissected from the mesencephalon at the level of the superior colliculus, where the central region of the pars compacta is located, while the head of the CN and the adjacent PU were dissected according to the NBB protocols. Samples from the SN pars compacta, CN and PU were collected and RNA was isolated as previously described (6).
RNA purity was determined using a NanoDrop ND‐1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RNA Integrity Number (RIN) was measured on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). All samples chosen in the study had RIN values of at least 6.2. (see Table S1). Above RIN = 5, the RIN does not affect qPCR quantification (24).
RNA of SN, CN and PU samples was reverse transcribed using 250 ng RNA, oligo dT primers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and 1/10 dilution of the total cDNA yield was used for all quantification reactions. Transcript quantifications were carried out on an ABI 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA). Each reaction was performed with 3 pmol of forward and reverse primers and 10 uL of 2x SYBR green reaction mix (Applied Biosystems) in a total volume of 20 µL. Sterile water and RNA samples without addition of reverse transcriptase in the cDNA synthesis were used as controls. The specificity of the amplification was checked by a melting curve analysis and electrophoresis of the products on an 8% polyacrylamide gel. Linearity of each qPCR assay was tested by measuring dilution series of the same stock cDNA on multiple plates. Reference genes were selected by a geNorm analysis (60) and used for normalization. For the SN, ACTB, MRPL24 and DHX16 were used; for the CN, GAPDH, PRPSAP1 and UFM1; while for the PU, ACTB, GAPDH, DHX16, GOT2 and FAM96B were used. The relative absolute amount of target genes was calculated by the formula 1010 × E−ct (E = 10−(1/slope)) (49). The absolute amount of transcript thus determined was then divided by the normalization factor to obtain the relative mRNA expression of the target gene.
Primer pairs, amplicon lengths and gene name abbreviation used in the text are given in Table S2.
IHC
IHC was performed on formalin fixed paraffin sections (6 µm thick) of human SN from the same PD patients and control subjects. Sections were mounted on SuperfrostPlus slides (Menzel, Germany) and dried overnight at 58°C followed by 24–36 h at 37°C. The sections were deparaffinized and rehydrated in an ethanol series and then rinsed in distilled water. After antigen retrieval treatment (0.01 M Citrate Buffer pH6 microwave for 10 minutes at 90°C), sections were allowed to cool for 30 minutes. After a blocking step with TBS/0.1% milk, a rabbit polyclonal anti‐steroid 5α‐R (1:500) was used as primary antibody (kindly provided by Prof D.W. Russell, University of Texas Southwestern Medical Center, Dallas, Texas, USA). A goat‐anti rabbit secondary antibody (1:400, Vector Laboratories, Peterborough, United Kingdom) was applied, followed by avidin biotin complex (ABC, 1:800, Vector Laboratories) and color developed with 0.5 mg/mL 3,3‐Diaminobenzidine‐tetrahydrochloride (Sigma, Zwijndrecht, the Netherlands) and ammonium nickel sulphate (2.2 mg/mL).
Statistical analysis
The Shapiro–Wilks test was used to test whether the data were normally distributed and performed in R (R Foundation for Statistical Computing software). Further statistical analysis was conducted with SPSS (version 16.0, SPSS Inc., Chicago, IL, USA). Log‐transformed gene expression values appeared to be normally distributed in most cases and therefore we used these for subsequent statistical analysis. Differences between groups were statistically evaluated by Student's t‐test followed by Benjamini and Hochberg's correction for multiple testing. Data from PD vs. control subjects were compared separately for SN, CN and PU. Values of P < 0.05 were considered significant. Fold‐changes were calculated using the median gene expression values.
RESULTS
Changes in neurosteroid biosynthetic pathways and GABA receptor gene expression in the SN
Transcript levels of two enzymes involved in the biosynthetic pathway of neurosteroids were found significantly different in the SN of PD patients compared with controls subjects. Levels of mRNA for 5α‐R1 and SULT2B1 were decreased in PD compared with controls by 1.6‐fold (P = 0.036) and 3.7‐fold (P = 0.036), respectively (1, 2). STS was found to have decreased 2.4‐fold while 3α‐HSD2 was increased 1.8‐fold in PD compared with controls but they were not significant after correction for multiple testing. The mRNA of the 5α‐reductase isoform 2 (5α‐R2) was not detected in the SN both in PD and in control subjects but was readily detected in liver positive controls (data not shown).
Figure 1.
5α ‐reductase changes in SN. A. Graph shows 5α–R1 significantly reduced gene expression in Parkinson's disease (PD) compared with controls (*P < 0.05). B–C. Photomicrographs of 5α–R immunostaining in substantia nigra of the pars compacta show reduced signal in the neurons (C) compared with controls (CTR) (B). Lines in the graphs represent median values. Full or open symbols represent, respectively, male or female subjects. Scale bar = 25 µm.
Figure 2.
Other gene expression changes in substantia nigra. Expression of SULT2B1 and some GABA‐A receptor subunits was significantly reduced Parkinson's disease (PD) compared with control (CTR) subjects: A. SULT2B1 (*P < 0.05); B. GABRA4 (*P < 0.05); C. GABRB1 (*P < 0.05).
Within the GABA and neurosteroid receptors, we found significantly reduced transcript expression for some subunits of the GABA‐A receptor as: GABRA4 was reduced 3.6‐fold (P = 0.036); GABRB1 was 1.8‐fold lower (P = 0.036) than controls (Figure 2B,C). GABRG2, GABRB2 and androgen receptor were, respectively, 2.5‐, 2.4‐ and 1.4‐fold reduced while GABRE was 7.4‐fold increased in PD compared with control but the significance was lost after multiple testing correction.
Gene expression changes of neurosteroid biosynthetic enzymes and GABA receptors in CN and in PU
In CN, we found 2.6‐fold increased gene expression of 3α‐HSD3 (P = 0.018) in PD patients compared with controls (Figure 3A). Within the receptors, the subunit GABRA4 mRNA was 22‐fold increased (P = 0.006) in PD subjects (Figure 3B). Increased mRNA levels were found for 3α‐HSD2, HSD17B4, HSD17B6, HSD17B7 and ER1 but these were not significant after correcting for multiple comparison.
Figure 3.
Gene expression changes in caudate nucleus (CN). Graphs show that 3α‐HSD3 (A) and GABRA4 (B) mRNA levels are significantly increased in CN of Parkinson's disease (PD) compared with controls (CTR) (*P < 0.05; **P < 0.01).
In PU, the mRNA levels of HSD17B6, HSD17B7, 3α‐HSD2 were found to have increased, respectively, by 1.4‐, 1.3‐ and 1.3‐fold while SULT2B1, GAD1, GABRA1, GABRB2, GABRA4, were found to have decreased, respectively, by 1.3‐, 2.2‐, 2‐, 1.7‐ and 3.4‐fold in PD compared with control, but these differences were lost after correction for multiple testing.
Statistical significance of the main qPCR results before and after the Benjamini and Hochberg correction in SN, CN and PU and the direction of gene expression changes are shown in Table S3.
IHC
Very strong staining for 5α‐R was present prevalently in pigmented neurons within the SN pars compacta of control patients, but moderate to strong staining was also present in several nonpigmented neurons of the SN pars reticulata. In PD patients, neuronal expression was evidently reduced especially in the pigmented neurons of the SN pars compacta (Figure 1B,C).
DISCUSSION
Biosynthetic enzyme changes
In this study, we investigated 37 genes involved in the synthesis of steroids or GABA neurotransmission in brain areas affected by PD.
In the SN of PD patients, two enzymes were downregulated. First, we found a downregulation of SULT2B1. This enzyme catalyzes the conversion of pregnenolone and dehydroepiandrosterone (DHEA) into their sulfated esters (ie, PS and DHEAS). No significant differences were found in the gene expression of the sulfatase which catalyzes the reverse reaction. The reduced expression of SULT2B1, if translated into reduced enzymatic activity, may indicate a reduced synthesis of PS and of DHEAS. This would also have implications for neurotransmission and neuroprotection; for example, PS is considered an excitatory neuroactive steroid for its negative modulation of the GABA‐A and AMPA receptors and its positive modulation of N‐methyl‐D‐aspartic acid (NMDA) receptors 33, 41, 55, 63, while DHEAS has been shown to promote neurogenesis, neurite growth and neuroprotective effects in rodents 34, 36, 58.
Second, we found a reduction in mRNA for 5α‐R1 in the SN of PD patients. IHC indicates that the protein is also reduced and the reduction in expression is found in both the pigmented and nonpigmented large neurons of the SN. The mRNA downregulation appears not to be solely a result of the loss of all large neurons in SN, because the magnitude of the change exceeds the degree of loss of these neurons, which was previously found to be 30% in these patients (6).
This finding correlates with the decreased levels for this rate‐limiting enzyme previously found in PMBC (40), suggesting that changes in steroid synthesis and metabolism may be systemically affected. A reduced amount of 5α‐R protein in SN neurons may lead to a reduced synthesis of 5α‐DHP and consequently of allopregnanolone. This idea is supported by our previous observation that these steroids were lower in the CSF of PD patients (19).
Allopregnanolone and its precursor 5α‐DHP have shown to protect neurons against NMDA and kainic acid induced excitotoxicity 14, 38, decrease apoptosis 20, 21, 32 and promote normal myelination and proliferation of neural progenitor cells 28, 54, 61, 62.These compounds may therefore have important neuroprotective roles and a decrease in their synthesis or metabolism may be a contributing factor to neuronal degeneration in SN.
In contrast, in the CN, mRNA levels of the enzyme 3α‐HSD3 were increased. This enzyme catalyzes the synthesis of the pregnane steroids allopregnanolone and THDOC. One may hypothesize that synthesis of steroids may vary in different brain areas according to the neurodegenerative process, which is known to affect SN and not CN (9). In this context, the increase of this neurosteroid synthetic enzyme may be part of a compensatory mechanism in CN in response to the loss of input from the SN. The downregulation in neurosteroid synthetic enzymes in the SN, on the other hand, appears to be related to the neuropathological process and in principle may play a causative role in the pathogenesis of the disease leading to a reduction in neuroprotection in SN.
GABA‐A receptor subunit changes
Major inputs to the SN come from the caudate–putamen complex and the pallidum and are mostly GABAergic (5). In this study, we found reduced mRNA levels of GABA‐A subunit α4 and β1 in SN while a 22‐fold increase of α4 was found in CN. This suggests that changes may occur in GABAergic transmission in the nigra. In contrast, no significant differences were found in the PU, suggesting that neurosteroid biosynthesis and GABAergic transmission in the PU are less affected in PD, compared with SN and CN.
There is increasing evidence that changes in peripheral and CNS concentration of steroids may play an important role in regulating the expression of various subunits of the GABA receptor 17, 25. In particular, GABRA4 subunit gene expression is influenced by progesterone and allopregnanolone fluctuation in different brain areas 2, 3.
The downregulation of GABRA4 subunit found in SN and its upregulation in CN could reflect a difference in pregnane steroid synthesis as suggested by differences in 5α‐R1 and 3α‐HSD3 gene expression, respectively, found in these areas. In this way, changes in pregnane steroid expression in SN may have complex effects on GABAergic neurotransmission, not only by direct modulation of the receptors but also by influencing expression of GABA‐A subunits.
Interestingly, the reduction in the SN in enzymes that synthesize allopregnanolone combined with reduced expression of GABA‐A receptor subunits hints that these changes may arise out of a possible compensatory mechanism in this brain area. Both these effects might be expected to lead to a reduction in GABAergic neurotransmission in the SN and consequently to increased dopamine release in the striatum.
In various studies, an acute reduction of progesterone or allopregnanolone levels was found to be associated with an increase in GABRA4 expression. This was seen following the sudden reduction of these steroids that occurs post‐partum or during the estrous cycle 16, 39, 53. Increased GABRA4 was also seen following withdrawal of progesterone or allopregnanolone after sustained administration, in rat hippocampus CA1 pyramidal cells 31, 56 and in cerebellum primary culture (25). In the present study, we observed, however, that reduced levels of 5α‐R1, which may indicate a reduced synthesis of allopregnanolone, are associated with decreased mRNA levels of GABRA4 subunit in the SN of PD patients. In the CN, increased expression of 3α‐HSD3, which may lead to higher synthesis of allopregnanolone, is coupled with an increased expression of GABRA4. Changes in neurosteroid synthesis in PD are most probably chronically protracted, prevailing for years in contrast to days or months in in vitro and in vivo animal experimental studies. In addition, the changes in GABRA4 synthesis in PD may reflect the long‐term response to a pathological process, as opposed to physiological conditions such as partum and the estrous cycle. Species and brain structure differences might also account for the seeming discrepancies in changes in GABRA4 expression and neurosteroid synthesis.
It is possible that chronic dopaminomimetic treatments received by the PD patient group could influence the results as these have been shown to sometimes affect GABA‐A receptors in the basal ganglia of PD 12, 27, 37, 48, 52. Despite the dramatic nigral denervation, an increased 37, 48 or unchanged 12, 27, 52 GABA concentration has been found in the caudate of PD patients in dopaminergic treatment before death. Therefore, the remarkably high (22‐fold) increase of the GABRA4 subunit mRNA expression found in the anterior part of caudate indicates a possible increase of GABA‐A receptors containing this subunit and which are known to be prevalently extrasynaptic and to mediate the tonic GABAergic inhibition (23).
Further work is necessary to determine if and how GABAergic transmission is affected by neurosteroids in the SN and CN.
This evidence suggests that there may be therapeutic opportunities for allopregnanolone in PD. To date, the potential clinical use of pregnane steroids has not yet been considered.
Allopregnanolone and its 3β‐methyl‐substituted analog (Ganaxolone) have been widely tested for their anxiolytic and antidepressant properties in preclinical studies, which have not shown severe side effects 35, 47. They are currently successfully used to treat catamenial epilepsy and a genetic neurodegenerative disorder involving dysregulation of neurosteroidogenesis (Niemann Pick type C disease) 13, 44, 50.
In the case of PD, if in vivo and in vitro studies accurately replicate the human situation, steroid replacement therapy may potentially be a useful treatment for this neurodegenerative disease.
This study presents new findings of changes in the regulation of neurosteroid synthesis and in the regulation of GABA‐A receptors, which suggest a role for pregnane steroids in PD.
Future directions
The changes in gene expression that we describe suggest that neurosteroid levels are likely to be affected during PD. Measurements of the compounds themselves in the SN are an important next step to build on these observations. In addition, it would be useful to study the neuroprotective effects of pregnane steroids in in vivo and in vitro models of PD.
FINANCIAL DISCLOSURES
None of the authors has reported biomedical financial interests or potential conflicts of interest in this work.
Supporting information
Table S1. Subjects clinicopathological information.
Table S2. List of genes studied and primers used for qPCR.
Table S3. Statistical significance of main qPCR results.
Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
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ACKNOWLEDGMENTS
The authors would like to thank, S. van de Bilt, R. Balesar, B. Fisser, A. Sluiter, U. Unmehopa, J.J. Van Heerikhuize for technical assistance and Dr M.R.J. Mason for statistical advises and for critical reading of the paper. We thank the Netherlands Brain Bank for providing the human material.
REFERENCES
- 1. Baulieu EE, Robel P, Schumacher M (2001) Neurosteroids: beginning of the story. Int Rev Neurobiol 46:1–32. [DOI] [PubMed] [Google Scholar]
- 2. Biggio F, Gorini G, Caria S, Murru L, Mostallino MC, Sanna E, Follesa P (2006) Plastic neuronal changes in GABA(A) receptor gene expression induced by progesterone metabolites: in vitro molecular and functional studies. Pharmacol Biochem Behav 84:545–554. [DOI] [PubMed] [Google Scholar]
- 3. Biggio G, Follesa P, Sanna E, Purdy RH, Concas A (2001) GABAA‐receptor plasticity during long‐term exposure to and withdrawal from progesterone. Int Rev Neurobiol 46:207–241. [DOI] [PubMed] [Google Scholar]
- 4. Bixo M, Andersson A, Winblad B, Purdy RH, Backstrom T (1997) Progesterone, 5alpha‐pregnane‐3,20‐dione and 3alpha‐hydroxy‐5alpha‐pregnane‐20‐one in specific regions of the human female brain in different endocrine states. Brain Res 764:173–178. [DOI] [PubMed] [Google Scholar]
- 5. Bolam JP, Smith Y (1990) The GABA and substance P input to dopaminergic neurones in the substantia nigra of the rat. Brain Res 529:57–78. [DOI] [PubMed] [Google Scholar]
- 6. Bossers K, Meerhoff G, Balesar R, Van Dongen JW, Kruse CG, Swaab DF, Verhaagen J (2009) Analysis of gene expression in Parkinson's disease: possible involvement of neurotrophic support and axon guidance in dopaminergic cell death. Brain Pathol 19:91–107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Bourque M, Dluzen DE, Di Paolo T (2009) Neuroprotective actions of sex steroids in Parkinson's disease. Front Neuroendocrinol 30:142–157. [DOI] [PubMed] [Google Scholar]
- 8. Braak H, Braak E (1995) Staging of Alzheimer's disease‐related neurofibrillary changes. Neurobiol Aging 16:271–278. [DOI] [PubMed] [Google Scholar]
- 9. Braak H, Del Tredici K, Rub U, De Vos RA, Jansen Steur EN, Braak E (2003) Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 24:197–211. [DOI] [PubMed] [Google Scholar]
- 10. Callier S, Morissette M, Grandbois M, Di Paolo T (2000) Stereospecific prevention by 17beta‐estradiol of MPTP‐induced dopamine depletion in mice. Synapse 37:245–251. [DOI] [PubMed] [Google Scholar]
- 11. Callier S, Morissette M, Grandbois M, Pelaprat D, Di Paolo T (2001) Neuroprotective properties of 17beta‐estradiol, progesterone, and raloxifene in MPTP C57Bl/6 mice. Synapse 41:131–138. [DOI] [PubMed] [Google Scholar]
- 12. Calon F, Morissette M, Rajput AH, Hornykiewicz O, Bedard PJ, Di Paolo T (2003) Changes of GABA receptors and dopamine turnover in the postmortem brains of parkinsonians with levodopa‐induced motor complications. Mov Disord 18:241–253. [DOI] [PubMed] [Google Scholar]
- 13. Carter RB, Wood PL, Wieland S, Hawkinson JE, Belelli D, Lambert JJ et al (1997) Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha‐hydroxy‐3beta‐methyl‐5alpha‐pregnan‐20‐one), a selective, high‐affinity, steroid modulator of the gamma‐aminobutyric acid(A) receptor. J Pharmacol Exp Ther 280:1284–1295. [PubMed] [Google Scholar]
- 14. Ciriza I, Azcoitia I, Garcia‐Segura LM (2004) Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo. J Neuroendocrinol 16:58–63. [DOI] [PubMed] [Google Scholar]
- 15. Compagnone NA, Mellon SH (2000) Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol 21:1–56. [DOI] [PubMed] [Google Scholar]
- 16. Concas A, Mostallino MC, Porcu P, Follesa P, Barbaccia ML, Trabucchi M et al (1998) Role of brain allopregnanolone in the plasticity of gamma‐aminobutyric acid type A receptor in rat brain during pregnancy and after delivery. Proc Natl Acad Sci U S A 95:13284–13289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Concas A, Follesa P, Barbaccia ML, Purdy RH, Biggio G (1999) Physiological modulation of GABA(A) receptor plasticity by progesterone metabolites. Eur J Pharmacol 375:225–235. [DOI] [PubMed] [Google Scholar]
- 18. D'Astous M, Morissette M, Tanguay B, Callier S, Di Paolo T (2003) Dehydroepiandrosterone (DHEA) such as 17beta‐estradiol prevents MPTP‐induced dopamine depletion in mice. Synapse 47:10–14. [DOI] [PubMed] [Google Scholar]
- 19. Di Michele F, Longone P, Romeo E, Lucchetti S, Brusa L, Pierantozzi M et al (2003) Decreased plasma and cerebrospinal fluid content of neuroactive steroids in Parkinson's disease. Neurol Sci 24:172–173. [DOI] [PubMed] [Google Scholar]
- 20. Djebaili M, Hoffman SW, Stein DG (2004) Allopregnanolone and progesterone decrease cell death and cognitive deficits after a contusion of the rat pre‐frontal cortex. Neuroscience 123:349–359. [DOI] [PubMed] [Google Scholar]
- 21. Djebaili M, Guo Q, Pettus EH, Hoffman SW, Stein DG (2005) The neurosteroids progesterone and allopregnanolone reduce cell death, gliosis, and functional deficits after traumatic brain injury in rats. J Neurotrauma 22:106–118. [DOI] [PubMed] [Google Scholar]
- 22. Do Rego JL, Seong JY, Burel D, Leprince J, Luu‐The V, Tsutsui K et al (2009) Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front Neuroendocrinol 30:259–301. [DOI] [PubMed] [Google Scholar]
- 23. Farrant M, Nusser Z (2005) Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci 6:215–229. [DOI] [PubMed] [Google Scholar]
- 24. Fleige S, Pfaffl MW (2006) RNA integrity and the effect on the real‐time qRT‐PCR performance. Mol Aspects Med 27:126–139. [DOI] [PubMed] [Google Scholar]
- 25. Follesa P, Serra M, Cagetti E, Pisu MG, Porta S, Floris S et al (2000) Allopregnanolone synthesis in cerebellar granule cells: roles in regulation of GABA(A) receptor expression and function during progesterone treatment and withdrawal. Mol Pharmacol 57:1262–1270. [PubMed] [Google Scholar]
- 26. Garcia‐Segura LM, Sanz A, Mendez P (2006) Cross‐talk between IGF‐I and estradiol in the brain: focus on neuroprotection. Neuroendocrinology 84:275–279. [DOI] [PubMed] [Google Scholar]
- 27. Gerlach M, Gsell W, Kornhuber J, Jellinger K, Krieger V, Pantucek F et al (1996) A post mortem study on neurochemical markers of dopaminergic, GABA‐ergic and glutamatergic neurons in basal ganglia‐thalamocortical circuits in Parkinson syndrome. Brain Res 741:142–152. [DOI] [PubMed] [Google Scholar]
- 28. Ghoumari AM, Ibanez C, El‐Etr M, Leclerc P, Eychenne B, O'Malley BW et al (2003) Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum. J Neurochem 86:848–859. [DOI] [PubMed] [Google Scholar]
- 29. Gillies GE, McArthur S (2010) Independent influences of sex steroids of systemic and central origin in a rat model of Parkinson's disease: a contribution to sex‐specific neuroprotection by estrogens. Horm Behav 57:23–34. [DOI] [PubMed] [Google Scholar]
- 30. Grandbois M, Morissette M, Callier S, Di Paolo T (2000) Ovarian steroids and raloxifene prevent MPTP‐induced dopamine depletion in mice. Neuroreport 11:343–346. [DOI] [PubMed] [Google Scholar]
- 31. Gulinello M, Gong QH, Li X, Smith SS (2001) Short‐term exposure to a neuroactive steroid increases alpha4 GABA(A) receptor subunit levels in association with increased anxiety in the female rat. Brain Res 910:55–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. He J, Hoffman SW, Stein DG (2004) Allopregnanolone, a progesterone metabolite, enhances behavioral recovery and decreases neuronal loss after traumatic brain injury. Restor Neurol Neurosci 22:19–31. [PubMed] [Google Scholar]
- 33. Horak M, Vlcek K, Petrovic M, Chodounska H, Vyklicky LJ (2004) Molecular mechanism of pregnenolone sulfate action at NR1/NR2B receptors. J Neurosci 24:10318–10325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Karishma KK, Herbert J (2002) Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons and prevents corticosterone‐induced suppression. Eur J Neurosci 16:445–453. [DOI] [PubMed] [Google Scholar]
- 35. Kask K, Backstrom T, Lundgren P, Sundstrom Poromaa I (2009) Allopregnanolone has no effect on startle response and prepulse inhibition of startle response in patients with premenstrual dysphoric disorder or healthy controls. Pharmacol Biochem Behav 92:608–613. [DOI] [PubMed] [Google Scholar]
- 36. Kimonides VG, Khatibi NH, Svendsen CN, Sofroniew MV, Herbert J (1998) Dehydroepiandrosterone (DHEA) and DHEA‐sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid‐induced neurotoxicity. Proc Natl Acad Sci U S A 95:1852–1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37. Kish SJ, Rajput A, Gilbert J, Rozdilsky B, Chang LJ, Shannak K, Hornykiewicz O (1986) Elevated gamma‐aminobutyric acid level in striatal but not extrastriatal brain regions in Parkinson's disease: correlation with striatal dopamine loss. Ann Neurol 20:26–31. [DOI] [PubMed] [Google Scholar]
- 38. Lockhart EM, Warner DS, Pearlstein RD, Penning DH, Mehrabani S, Boustany RM (2002) Allopregnanolone attenuates N‐methyl‐D‐aspartate‐induced excitotoxicity and apoptosis in the human NT2 cell line in culture. Neurosci Lett 328:33–36. [DOI] [PubMed] [Google Scholar]
- 39. Lovick TA, Griffiths JL, Dunn SM, Martin IL (2005) Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats. Neuroscience 131:397–405. [DOI] [PubMed] [Google Scholar]
- 40. Luchetti S, Di MF, Romeo E, Brusa L, Bernardi G, Cummings BJ, Longone P (2006) Comparative non‐radioactive RT‐PCR assay: an approach to study the neurosteroids biosynthetic pathway in humans. J Neurosci Methods 153:290–298. [DOI] [PubMed] [Google Scholar]
- 41. Majewska MD, Schwartz RD (1987) Pregnenolone‐sulfate: an endogenous antagonist of the gamma‐aminobutyric acid receptor complex in brain? Brain Res 404:355–360. [DOI] [PubMed] [Google Scholar]
- 42. Majewska MD, Harrison NL, Schwartz RD, Barker JL, Paul SM (1986) Steroid hormone metabolites are barbiturate‐like modulators of the GABA receptor. Science 232:1004–1007. [DOI] [PubMed] [Google Scholar]
- 43. Melcangi RC, Garcia‐Segura LM, Mensah‐Nyagan AG (2008) Neuroactive steroids: state of the art and new perspectives. Cell Mol Life Sci 65:777–797. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44. Mellon SH, Gong W, Schonemann MD (2008) Endogenous and synthetic neurosteroids in treatment of Niemann‐Pick type C disease. Brain Res Rev 57:410–420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Morissette M, Al SS, Callier S, Di Paolo T (2008) Estrogen and SERM neuroprotection in animal models of Parkinson's disease. Mol Cell Endocrinol 290:60–69. [DOI] [PubMed] [Google Scholar]
- 46. Morita K, Her S (2008) Progesterone pretreatment enhances serotonin‐stimulated BDNF gene expression in rat c6 glioma cells through production of 5alpha‐reduced neurosteroids. J Mol Neurosci 34:193–200. [DOI] [PubMed] [Google Scholar]
- 47. Nohria V, Giller E (2007) Ganaxolone. Neurotherapeutics 4:102–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48. Perry TL, Javoy‐Agid F, Agid Y, Fibiger HC (1983) Striatal GABAergic neuronal activity is not reduced in Parkinson's disease. J Neurochem 40:1120–1123. [DOI] [PubMed] [Google Scholar]
- 49. Pfaffl MW (2001) A new mathematical model for relative quantification in real‐time RT‐PCR. Nucleic Acids Res 29:e45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Pieribone VA, Tsai J, Soufflet C, Rey E, Shaw K, Giller E, Dulac O (2007) Clinical evaluation of ganaxolone in pediatric and adolescent patients with refractory epilepsy. Epilepsia 48:1870–1874. [DOI] [PubMed] [Google Scholar]
- 51. Ramirez AD, Liu X, Menniti FS (2003) Repeated estradiol treatment prevents MPTP‐induced dopamine depletion in male mice. Neuroendocrinology 77:223–231. [DOI] [PubMed] [Google Scholar]
- 52. Rinne JO, Halonen T, Riekkinen PJ, Rinne UK (1988) Free amino acids in the brain of patients with Parkinson's disease. Neurosci Lett 94:182–186. [DOI] [PubMed] [Google Scholar]
- 53. Sanna E, Mostallino MC, Murru L, Carta M, Talani G, Zucca S et al (2009) Changes in expression and function of extrasynaptic GABAA receptors in the rat hippocampus during pregnancy and after delivery. J Neurosci 29:1755–1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54. Schumacher M, Guennoun R, Stein DG, De Nicola AF (2007) Progesterone: therapeutic opportunities for neuroprotection and myelin repair. Pharmacol Ther 116:77–106. [DOI] [PubMed] [Google Scholar]
- 55. Shirakawa H, Katsuki H, Kume T, Kaneko S, Akaike A (2005) Pregnenolone sulphate attenuates AMPA cytotoxicity on rat cortical neurons. Eur J Neurosci 21:2329–2335. [DOI] [PubMed] [Google Scholar]
- 56. Smith SS, Gong QH, Hsu FC, Markowitz RS, Ffrench‐Mullen JM, Li X (1998) GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature 392:926–930. [DOI] [PubMed] [Google Scholar]
- 57. Stoffel‐Wagner B (2001) Neurosteroid metabolism in the human brain. Eur J Endocrinol 145:669–679. [DOI] [PubMed] [Google Scholar]
- 58. Suzuki M, Wright LS, Marwah P, Lardy HA, Svendsen CN (2004) Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex. Proc Natl Acad Sci U S A 101:3202–3207. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59. Tomita H, Vawter MP, Walsh DM, Evans SJ, Choudary PV, Li J et al (2004) Effect of agonal and postmortem factors on gene expression profile: quality control in microarray analyses of postmortem human brain. Biol Psychiatry 55:346–352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 60. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van RN, De Paepe A, Speleman F (2002) Accurate normalization of real‐time quantitative RT‐PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034.1‐0034.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 61. Wang JM, Johnston PB, Ball BG, Brinton RD (2005) The neurosteroid allopregnanolone promotes proliferation of rodent and human neural progenitor cells and regulates cell‐cycle gene and protein expression. J Neurosci 25:4706–4718. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62. Wang JM, Irwin RW, Liu L, Chen S, Brinton RD (2007) Regeneration in a degenerating brain: potential of allopregnanolone as a neuroregenerative agent. Curr Alzheimer Res 4:510–517. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63. Wu FS, Gibbs TT, Farb DH (1991) Pregnenolone sulfate: a positive allosteric modulator at the N‐methyl‐D‐aspartate receptor. Mol Pharmacol 40:333–336. [PubMed] [Google Scholar]
Associated Data
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Supplementary Materials
Table S1. Subjects clinicopathological information.
Table S2. List of genes studied and primers used for qPCR.
Table S3. Statistical significance of main qPCR results.
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