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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Psychoneuroendocrinology. 2018 Feb 7;90:43–51. doi: 10.1016/j.psyneuen.2018.02.004

Pregnenolone-progesterone-allopregnanolone pathway as a potential therapeutic target in first-episode antipsychotic-naïve patients with schizophrenia

HuaLin Cai a,b,c, Xiang Zhou a,b, George G Dougherty a,d, Ravinder D Reddy e, Gretchen L Haas a,d, Debra M Montrose d, Matcheri Keshavan d,f, Jeffrey K Yao a,b,d,*
PMCID: PMC5864547  NIHMSID: NIHMS941916  PMID: 29433072

Abstract

Neurosteroids are both endogenous and exogenous steroids that rapidly alter neuronal excitability through interactions with ligand-gated ion channels and other cell surface receptors. They are originated from cholesterol and have important implications for schizophrenia (SZ) pathophysiology and treatment strategies. Specifically, pregnenolone (PREG), progesterone (PROG) and allopregnanolone (ALLO) exhibit similar psychotropic properties. Using enzyme immunoassay, we compared the neurosteroids in PREG downstream pathways in plasma between healthy controls (HC, n=43) and first-episode antipsychotic-naïve patients with SZ (FEAN-SZ, n=53) before antipsychotic drug (APD) treatment. Comparisons were also made particularly along PREG-PROG-ALLO pathway in the same FEAN-SZ patients across multiple time points following initiation of treatment for 12 months (m). Firstly, at baseline, levels of PREG were significantly higher and those of ALLO were lower in FEAN-SZ than in HC, whereas PROG, cortisol, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were not different. Consequently, the molar ratios of ALLO/PREG and ALLO/PROG in FEAN-SZ were significantly reduced. Secondly, in response to APD at 1 month, ALLO levels in FEAN-SZ were markedly elevated, whereas PREG and PROG levels decreased. Thirdly, among FEAN-SZ, lower levels of PROG (reflecting higher conversion to ALLO) at baseline may predict better therapeutic outcome after 1 month of APD treatment. These findings point to the perturbations of the PREG-PROG-ALLO pathway early in psychosis, and further study of this pathway may inform alternative and innovative therapeutic targets for SZ.

1. Introduction

Neuroactive steroids are both endogenous and exogenous steroids, which can alter brain excitability by binding to ligand-gated ion channels as well as cell surface receptors, e.g., γ-aminobutyric acidA (GABAA) receptor (Lan and Gee, 1994). They are synthesized from cholesterol, which is converted into pregnenolone (PREG) and then into other endogenous steroids (Fig. 1). Although locally synthesized neurosteroids play a major role as signaling molecules in the brain, the mechanisms underlying the regulation of neurosteroid biosynthesis remain to be elucidated. Since neurosteroids are highly lipophilic and readily taken up across the blood-brain barrier, those neurosteroids and their precursors that are produced by an endocrine gland in the periphery can also exert their biological functions by subsequently reaching the brain through the bloodstream. A previous study (Mensah-Nyagan et al., 1999) indicated that the expression of several key steroidogenic enzymes in the brain may be regulated by adrenal and gonadal steroids, suggesting a putative association between peripheral and brain neurosteroids. Furthermore, their findings suggest that neurosteroids in the periphery may serve as a proxy or surrogate marker utility for regulation of neurosteroids in the brain. In fact, positive correlations between plasma and brain levels of neurosteroids have been demonstrated in rats (Barbaccia et al., 1997, 2001). A similar correlation has also been observed between plasma and CSF in humans (Kim et al., 2000). Among the neurosteroids, PREG, progesterone (PROG), allopregnanolone (ALLO), cortisol (CORT), and dehydroepiandrosterone (DHEA) are involved in different aspects of biological functions that have been implicated in the pathophysiology of some psychiatric disorders.

Fig. 1.

Fig. 1

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Pregnenolone downstream pathways. Metabolites identified by colored background were determined in the present study. Abbreviations: DH, dihydro; 1, cytochrome P450scc enzyme (or CYP11A1); 2, 3β-hydroxysteroid dehydrogenase; 3, 5α-reductase; 4, 3α-hydroxysteroid dehydrogenase; 5, 17α-hydroxylase; 6, 21β-hydroxylase; 7, 11β-hydroxylase; 8, 17,20 lyase; 9, sulfotransferase; 10, sulfatase; 11, 3β-hydroxysteroid dehydrogenase 2/Δ5–4 isomerase.

Converging evidence indicates a pattern of reduced PREG and its related endogenous steroids in schizophrenia (SZ). Low levels of serum PREG have been found in SZ patients compared to healthy controls (HC) (Ritsner et al., 2007). Levels of plasma CORT in male SZ patients with moderate negative symptoms were significantly higher than HC (Shirayama et al., 2002). Also, significant correlations between levels of plasma CORT and severity of negative symptom were observed in male SZ patients (Shirayama et al., 2002), suggesting that this endogenous steroid may serve as a biological marker for the severity of negative symptoms in SZ patients. In another study, following metabolic stress induced by injection of 2-deoxyglucose, SZ patients exhibited a significantly greater increase in plasma PROG compared to HC (Breier and Buchanan, 1992).

Moreover, findings from a proof-of-concept trial with adjunctive PREG treatment suggested that increases in serum PREG and ALLO levels may predict cognitive outcome after 8 weeks (Marx et al., 2009). In a recent follow-up clinical trial, this same group of investigators further concluded that PREG improved functional capacity in SZ patients, but did not improve cognitive symptoms after 8 weeks of treatment, suggesting that neurosteroids offer partial promise as a new therapeutic agent for SZ (Marx et al., 2014).

In several pharmacologic interventions in rodents, increases of PROG and ALLO levels in the brain were observed after clozapine and haloperidol administration (Khisti et al., 2002); interestingly, intracerebroventricular administration of ALLO produced inhibition of amphetamine-induced motor hyperactivity (Khisti et al., 2002). Progesterone and ALLO administered intraperitoneally and intracerebro-ventricularly, respectively, also inhibited the conditioned avoidance response in rodents (Ugale et al., 2004), an effect that was also observed following intraperitoneal injection of olanzapine, risperidone, and haloperidol (Ugale et al., 2004; Wadenberg et al., 2001). These psychotropic-like effects of progesterone and ALLO are likely due to interactions between these steroids, the GABAergic system and the dopaminergic system (Jaworska-Feil et al., 1998; Lewis et al., 2004).

Thus far, the bulk of the evidence for the psychotropic-like effects of neuroactive steroids like PROG and ALLO has come from animal models or human studies of antipsychotic drug (APD)-treated patients with chronic SZ. We hypothesize that 1) a homeostatic imbalance in PREG-PROG-ALLO pathway exists early in the course of SZ, and 2) the PREG-PROG-ALLO pathway may be a therapeutic target for antipsychotic treatment. To address these hypotheses in the present study, we compared six key plasma neurosteroids including PREG, PROG, ALLO, CORT, DHEA, and DHEA sulfate (DHEAS) between HC and first-episode antipsychotic-naïve patients with schizophrenia (FEAN-SZ) before initiation of APD treatment (baseline). Comparisons of parameters at baseline were also made in the same FEAN-SZ individuals across multiple time points following APD treatment.

2. Materials and methods

2.1. Participants

Blood samples were obtained after overnight fasting from 53 patients who were recruited in their first-episode of psychosis after they met DSM-IV criteria for SZ, schizophreniform or schizoaffective disorder based on the Structured Clinical Interview for DSM Diagnosis (SCID), and 43 age- and gender-matched HC subjects drawn from the same communities as where the patients were recruited. The follow-up blood samples were also obtained in the same patient individuals at 1, 6 and 12 months (m) after initiation of treatment with one or more of the following antipsychotic drugs: risperidone, olanzapine, quetiapine, aripiprazole and haloperidol. Approximately, 81% of patients were treated with risperidone either as a single drug (74%) or as an adjunctive (26%).

2.2. Clinical assessments

Results of the initial diagnostic assessments were reviewed and final research diagnoses were determined at consensus diagnostic conferences reviewing SCID and all clinical data, attended by research faculty and staff, and chaired by one of the authors (MSK or DM). All subjects provided signed informed consent after a full explanation of the study. The study was approved by the Institutional Review Boards of both the University of Pittsburgh and the VA Pittsburgh Healthcare System. Clinical symptomatology and global clinical status were rated by experienced research clinicians at all time points using the Brief Psychiatric Rating Scales (BPRS; Overall and Gorham, 1962), the Schedules for the Assessment of Positive (SAPS) and Negative Symptoms (SANS) (Andreasen and Olsen, 1982), the Global Assessment Scale (GAS; Endicott et al., 1976); the Hamilton Depression Rating Scale (HDRS; Williams, 1988), and the Clinical Global Impression (CGI) severity scale (Guy, 1976).

2.3 Plasma neurosteroid assays

To reduce the variance associated with circadian rhythm effects (Brambilla et al., 2009), blood samples were drawn around 8:00 am on the morning of each session. In addition, since the steroid hormones can change drastically throughout the menstrual cycle, for female subjects, we inquired about the date of their last menstrual bleeding and restricted the sampling time to the period within the follicular phase (Day 1–13). Briefly, freshly drawn blood with anticoagulant citrate dextrose was centrifuged at 750×g for 7 minutes to remove RBC and stored at −80 °C. Aliquots of banked samples that have not previously thawed were used for assay.

Plasma neurosteroid concentrations were analyzed in duplicate by quantitative sandwich enzyme linked immunoassay (ELISA) using commercially available kits. Pregnenolone and DHEAS were purchased from BioVender (Modrice, Czech Republic). Dehydroepiandrosterone was acquired from Eagle Biosciences (Nashua, NH, USA). Progesterone and CORT were provided by Cayman Chemical (Ann Arbor, MI, USA). Allopregnanolone was obtained from Arbor Assays (Ann Arbor, MI, USA) and its original sensitivity was 0.13 ng/mL as indicated in the brochure. We speculated that this sensitivity may not be feasible for some plasma samples with lower concentrations. To increase the sensitivity for the determination of ALLO, 500 μL of plasma was extracted by ethyl acetate, evaporated to dryness and dissolved in 100 μL of assay buffer, which means that the sample was concentrated five times before ELISA analysis. The sensitivity, cross-reactivity, intra- and inter-assay CVs in the brochures among different ELISA kits are summarized in Supplementary Table 1.

2.4. Statistical Analyses

Data, per plan, consisted of 6 steroid hormone concentrations (PREG, PROG, ALLO, CORT, DHEA, DHEAS), and covariates [gender, age, body mass index (BMI)], measured for 43 HC and 53 FEAN-SZ, with the latter group having data at baseline, and after 1, 6, and 12 months of antipsychotic treatment. Each analyte was measured by a separate plasma aliquot. Since earlier studies had used up portions of the samples, some subjects were left with fewer than the required number of aliquots for all analytes. However, all assignments of aliquots to particular analyte measurements were made prior to observing any outcomes of the determinations. None of the aliquots measured yielded results deemed invalid or undetectably low. The patterns of obtained data are presented in Tables 1 and 2. All analyses were performed using S-Plus v. 8.1.1 software.

Table 1.

Descriptive statistics of metabolites in pregnenolone downstream pathway.

Groups Mean* SD Median Firstqrt Thirdqrt Student t test p† (HC vs FEAN-SZ)
1. HC
 Pregnenolone 1.430 1.373 0.970 0.400 1.920
 Progesterone 0.533 0.113 0.540 0.448 0.595
 Allopregnanolone 1.032 0.426 1.015 0.685 1.268
 Cortisol 90 56 76 45 142
 DHEA 6.676 3.146 7.830 3.970 9.250
 DHEA sulfate 1433 592 1441 991 1892
2. FEAN-SZ-BL
 Pregnenolone 3.242 2.618 2.550 1.218 4.898 − 3.704 0.0005
 Progesterone 0.557 0.103 0.575 0.490 0.618 − 0.687 0.3890
 Allopregnanolone 0.679 0.468 0.525 0.373 0.760 3.779 0.0004
 Cortisol 147 192 87 53 127 − 1.011 0.3160
 DHEA 8.207 2.522 8.150 6.270 10.650 − 2.058 ‡ 0.0400
 DHEA sulfate 1693 733 1663 1145 2186 − 1.601 0.1140
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*

All values were expressed as ng/mL. The comparisons were made for pregnenolone, allopregnanolone and cortisol after their concentration values were ln-transformed for normality.

†

Significance with p <0.05/6 = 0.0083 after liberal Bonferroni correction of alpha.

‡

DHEA values are not normally distributed. The Z-statistic of the approximate Wilcoxon Rank Sum Test is displayed, for comparisons between HC and FEAN-SZ groups.

Abbreviations: SD, standard deviation; Firstqrt, first quartile; Thirdqrt, third quartile; HC, healthy controls; FEAN-SZ, first-episode antipsychotic-naïve patients with schizophrenia; BL, baseline; DHEA, dehydroepiandrosterone.

Table 2.

Molar ratios of product to precursor in the baseline PREG-PROG-ALLO pathway.

Groups Mean* SD Median Firstqrt Thirdqrt Student t test p‡ (HC vs FEAN-SZ)
1. HC
 PROG/PREG 0.869 1.042 0.427 0.257 0.943
 ALLO/PROG 1.995 0.845 1.879 1.392 2.725
 ALLO/PREG 1.760 2.668 0.805 0.435 1.519
 CORT/PROG 151 102 123 71 229
 CORT/PREG 98 88 72 27 158
 DHEA/PREG 16.047 20.473 7.414 2.372 23.202
 DHEAS/DHEA 241 174 173 116 367
 DHEAS/PREG 2212 3578 1066 469 2131
2. FEAN-SZ-BL
 PROG/PREG 0.404 0.524 0.217 0.109 0.456 2.555 0.0140
 ALLO/PROG 1.164 0.736 0.962 0.603 1.414 1080† 0.0001
 ALLO/PREG 0.475 0.600 0.167 0.104 0.668 693† 0.0006
 CORT/PROG 226 262 142 79 211 −0.996 0.3230
 CORT/PREG 62 87 29 14 68 2.480 0.0163
 DHEA/PREG 4.829 3.676 3.202 1.981 7.803 2.124 0.0397
 DHEAS/DHEA 165 73 152 106 214 1.265 0.2140
 DHEAS/PREG 844 873 568 298 1190 2.502 0.0162
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*

The ratios PROG/PREG, CORT/PROG, CORT/PREG, DHEA/PREG, DHEAS/DHEA and DHEAS/PREG were ln-transformed for normality before comparisons.

†

W statistics of the exact Wilcoxon Rank Sum test are displayed for comparisons of ratios ALLO/PROG or ALLO/PREG between HC and FEAN-SZ-BL groups.

‡

p < 0.05/8 = 0.00625 after liberal Bonferroni correction of alpha.

Abbreviations: SD, standard deviation; Firstqrt, first quartile; thirdqrt, third quartile; HC, healthy control subjects; FEAN-SZ, first-episode antipsychotic-naïve patients with schizophrenia; BL, baseline; PREG, pregnenolone, PROG, progesterone; ALLO, allopregnanolone, CORT, cortisol; DHEA, dehydroepiandrosterone; and DHEAS, dehydroepiandrosterone sulfate.

To begin, quantile-quantile plots and a correlation-type univariate normality test were used to assess analytes and selected ratios of them, within subject groups, for approximate normality (Johnson and Wichern, 1998). If both groups’ data were not approximately normally distributed, then nonparametric tests were used.

The distribution of covariates was checked for imbalances between the HC and FEAN-SZ groups using descriptive statistics. Dependence of the outcome concentration variables upon covariates was checked by Kendall’s tau correlations for age and BMI and t-tests of gender subgroup differences in the analytes within each of the HC and FEAN-SZ groups. These relationships were liberally accepted as significant if p < 0.05, uncorrected, so as not to miss any confounding effects.

For control of Type I error, the Bonferroni correction was applied to α = 0.05 separately for 4 sets of hypotheses of (i) group differences in original analytes at baseline; (ii) tests of baseline values vs. those at 1-, 6- and 12-m; (iii) tests of group differences in analyte ratios at baseline; and (iv) tests of baseline analyte ratio values vs. those at 1-, 6-, and 12-m.

Analytes were compared between HC and FEAN-SZ groups at baseline by Welch-modified (Welch, 1947) or standard t-tests, depending on variance inequality or equality, respectively, or (if not normally distributed) by approximate or exact Wilcoxon Rank Sum tests, depending on the presence or absence of ties, respectively (Lehmann, 1975). Analyte molar ratios composed of product-over-precursor, each concentration in nmol/mL (PROG/PREG, ALLO/PROG, ALLO/PREG, CORT/PROG, CORT/PREG, DHEA/PREG, DHEAS/DHEA, DHEAS/PREG), were also assessed for normality, and transformed as indicated above. Ratios were compared between groups by the same t- and Wilcoxon tests as were the original analytes.

Change in both analyte concentrations and ratios were tested within the FEAN-SZ group, by comparing values at 1, 6, and 12 months versus baseline, using paired t-tests or Wilcoxon Signed Ranks tests (WSRT). Again, exact or approximate Wilcoxon Rank Signed Ranks tests were used, depending on the absence or presence of ties, respectively (Lehmann, 1975).

When the FEAN-SZ group showed a significant difference in the analyte from baseline to follow-up, we then explored correlations of clinical ratings of symptom severity (BPRS, SAPS and SANS) and global clinical status (GAS and CGI severity) with the analyte concentrations at baseline and at each of the follow-up points. Because the ratings were not approximately normal at all time points, nor transformable to normality, the correlations were tested using Kendall’s tau. Uncorrected p-values are presented for this exploratory work.

3. Results

3.1. General descriptive statistics

Baseline characteristics of the subjects enrolled were essentially equivalent for the two groups: (1) mean age was 25.6 (± 7.2) years for HC and 24.9 (± 8.6) years for the FEAN-SZ group; (2) male/female ratios were 24/19 (HC) and 30/23 (FEAN-SZ); and (3) a slightly lower BMI was observed in the FEAN-SZ group (23.9 ± 5.4) as compare with the HC group (25.4 ± 4.6), as expected (Verma et al., 2009). It is noteworthy that GAS scores for FEAN-SZ subjects improved after 1 month treatment with antipsychotic drug (42.2±14.3 at baseline versus 31.7±13.0 at 1 month) (t = 4.648, df = 21, p = 0.0001).

We checked for associations between covariates (age, BMI, and gender), and the 6 concentration variables within the two groups. CORT(ln) was associated with age in HC only and with BMI in FEAN-SZ only. No significant difference was found between HC and FEAN-SZ groups for CORT (see below). In addition, there were no significant differences in our targeted plasma neurosteroids between male and female subjects (Supplementary Table 2).

3.2. Comparison of neurosteroids between HC and baseline FEAN-SZ

The normal ranges of plasma-targeted neurosteroids in the present study were comparable to those previously reported using either ELISA or Mass Spectroscopy assays (Supplementary Table 3). We also tested the levels of total cholesterol, a precursor of neurosteroids in plasma, which showed no significant differences between FEAN-SZ (154.2±33.0 mg/dL) and HC (157.9±34.2 mg/dL).

Since targeted plasma neurosteroids did not differ between male and female subjects (Supplementary Table 2), group differences in plasma neurosteroids were not adjusted for gender. Of the 6 steroid concentration variables (Table 1), only PREG and ALLO showed significant group differences, with mean PREG being higher and mean ALLO being lower in FEAN-SZ compared to HC.

A metabolic pathway generally consists of sequential biochemical reactions that generate various products from a set of precursors. Thus, connections between biochemical reactions via substrate and product metabolites form complex metabolic networks that may be analyzed using network theory, stoichiometric analysis, and information on protein structure/function and metabolite properties (Hatzimanikatis et al., 2004). Thus, estimates of various enzyme activities in the PREG downstream pathways were calculated using the product-to-precursor ratios for each enzyme (Fig. 1). Of the 8 molar ratios considered, only ALLO/PROG and ALLO/PREG showed significant group differences (Table 2). Both ratios were lower in FEAN-SZ than in HC subjects.

3.3. Effect of antipsychotic treatment on PREG-PROG-ALLO pathway

As illustrated in Table 1, only PREG (In) and ALLO (ln) showed significant group differences. Thus, the PREG-PROG-ALLO pathway (Fig. 1) was specifically investigated in response to APD treatment at 1, 6 and 12 months. All 3 neurosteroids, PREG, PROG and ALLO, showed significant changes from baseline to 1 month (Fig. 2A–2C). In addition, PREG (In) also showed a significant change from baseline to 6 months (Fig. 2A). Specifically, there was a robust increase of plasma ALLO levels (Fig. 2C) with a relative decrease of PREG (Fig. 2A) and PROG (Fig. 2B) levels in FEAN-SZ subjects after 1 month APD treatment. However, after 12 months of treatment, such differences from baseline were no longer present.

Fig. 2.

Fig. 2

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Effects of antipsychotic drug treatment on neurosteroid pregnenolone (A) and its metabolites progesterone (B) and allopregnanolone (C) concentrations as well as changes in molar ratios of PROG/PREG (D), ALLO/PROG (E) and ALLO/PREG (F) in first-episode antipsychotic-naïve patients with schizophrenia (FEAN-SZ) at baseline, 1-, 6- and 12- months (m). Column bar graphs and box-and whisker plots were used to depict the changes of neurosteroid concentrations and relevant molar ratios, respectively. For the column bar graphs (A, B and C), the top of the column and the error bar indicate the mean and the standard deviation, respectively. For the box-and whisker plots (D, E and F), the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The spread of the whiskers represents the minimum to maximum of all of the data points. P-values (uncorrected) are for comparisons of each of the 1-, 6-, and 12-m values with the corresponding baseline values, using Wilcoxon Signed Ranks Tests, due to non-normality of data at some time points. Any values exceeded mean±2SD were considered as outliers and excluded from the final boxplots.

Three molar ratios (PROG/PREG, ALLO/PROG, and ALLO/PREG) were selected to reflect the product and substrate relationships longitudinally as described in the PREG-PROG-ALLO pathway (Fig. 1). As shown in Fig. 2D–2F, all 3 ratios were significantly increased at 1 month as compared to baseline. Significant differences from baseline were also seen for ln(PROG/PREG) and ALLO/PREG after 6 months of treatment. However, there were no significant differences in molar ratios after 12 months of treatment.

3.4. Correlations between biochemical measures and clinical assessments

Of the 53 patients enrolled, 22 completed the 12 months duration of observation. As expected, the patients showed improvement in clinical symptoms and clinical functioning after 4 weeks of treatment with APD (Condray et al., 2011).

Exploratory testing was done for correlations between clinical ratings (sum of SANS Global scores sum of SANS Item scores, sum of SAPS Global scores, sum of SAPS Item scores, BPRS 18-item Total Score, BPRS Hallucination score, BPRS positive symptom score, BPRS negative symptom score, GAS, CGI severity score, and Hamilton Depression Rating Scale [HDRS] 17-item total score both at Baseline and at Week 4, and, on the other hand, plasma concentrations of PROG, ALLO, and PREG, also both at Baseline and Week 4. Thus a total of 156 correlation tests were run using Kendall’s tau (due to non-normality of some variables). Very few correlations reached significance (probability <0.05), and only one, PROG at BL vs. GAS at Week 4 reached p = 0.01 (Fig. 3, tau = −0.39). The correlation between change scores for GAS and PROG was also conducted, i.e. PROG at Week 4 minus PROG at BL vs. GAS at Week 4 minus GAS at BL was significant [tau = 0.43, p = 0.0046]. Should this result be seen again on replication, it would suggest that lower baseline levels of progesterone predict a higher GAS after 4 weeks of treatment, and that greater increases in progesterone with treatment are associated with greater improvements in global functioning, as measured on the GAS.

Fig. 3.

Fig. 3

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Correlations between plasma levels of neurosteroids [A, pregnenolone (PREG), B, progesterone (PROG) and C, allopregnanolone (ALLO)] at baseline and global assessment scale (GAS) in first-episode antipsychotic-naïve patients with schizophrenia (FEAN-SZ) after 1-month treatment with antipsychotic drugs (APD). Because the ratings were not approximately normal at all time points, nor transformable to normality with the same transformation, the correlations were estimated and tested using Kendall’s tau. Uncorrected p-values without a significance criterion are presented for this exploratory work.

4. Discussion

Several key findings emerged in the present study. Firstly, FEAN-SZ patients had significantly lower levels of plasma ALLO and higher levels of PREG (the precursor) than in HC (Table 1), whereas levels of PROG, CORT, DHEA and DHEAS were essentially the same. Consequently, there were significant reductions of molar ratios of PROG/PREG, ALLO/PROG and ALLO/PREG (Table 2), consistent with a decreased production of ALLO and an increased level of PREG in the FEAN-SZ patients before initiation of APD treatment. Secondly, following 4 weeks of APD treatment, plasma levels of ALLO were markedly elevated whereas levels of PROG and PREG (precursors) were relatively reduced in the FEAN-SZ patients (Fig. 2), suggesting a trend toward normalization in the metabolic conversion to ALLO with treatment. Thirdly, FEAN-SZ with lower levels of PROG (implying higher metabolic conversion to ALLO) at baseline may predict a better treatment outcome (Fig. 3B). Taken together, these findings point to the perturbations of the PREG-PROG-ALLO pathway early in psychosis and prior to treatment. Further investigation into this pathway may inform alternative and innovative therapeutic targets for SZ.

4.1. Homeostatic imbalance of PREG-PROG-ALLO pathway in schizophrenia

Our present findings that indicate a significantly reduced conversion of PREG to ALLO in the FEAN-SZ patients are in accordance with a preliminary study, showing reduced levels of plasma ALLO in non-medicated first-episode SZ patients (MacKenzie et al., 2007), and a post-mortem study demonstrating decreased ALLO levels in parietal cortex from SZ patients compared to HC (Marx et al., 2006a).

During the PROG catabolism, conversion from 5α-dehydroPROG to ALLO is readily reversible (Fig. 1). Since the activity of the 3α-hydroxysteroid dehydrogenase (3α-HSD) is far greater than that of the 5α-reductase, steroid 5α-reduction is thus the rate-limiting step in the biosynthesis from PROG to ALLO (Do Rego et al., 2009). Previous data have indicated that levels of ALLO and 5α-reductase in nervous tissues may be altered in SZ (Paba et al., 2011).

4.2. Neuroprotective roles of neurosteroids

Emerging preclinical and clinical evidence suggests that PREG may be a promising novel therapeutic candidate in schizophrenia (Marx et al., 2011). Pregnenolone administration results in elevations in downstream neurosteroids such as PROG and ALLO, which have multiple neuroprotective effects that may potentially be increased with therapeutic benefits (Marx et al., 2011). Specifically, PROG has a wide range of neuroprotective actions involving reductions of brain edema (Guo et al., 2006), inflammation and oxidant activity (He et al., 2004; Djebaili et al., 2005), maintaining mitochondrial functions (Robertson et al., 2006), regulating hemostatic proteins (VanLandingham et al., 2008), and promoting the survival of newborn neurons (Zhang et al., 2010), but not clinical benefit in patients with severe traumatic brain injury (Skolnick et al., 2014). Interestingly, ALLO originated from PROG can also mimic many of the neuroprotective actions associated with PROG (He et al., 2004; Djebaili et al., 2004; Sayeed et al., 2009).

In contrast to its precursor PROG, ALLO does not bind to the classical intracellular PROG receptors. ALLO is a potent endogenous neurosteroid that markedly potentiates the responses of GABAA receptor, the principal mediator of the fast inhibitory transmission within the central nervous system. Recently, it has been shown that the modulation of GABAA receptors by ALLO influences the synaptic release and activity of glycine, a major inhibitory neurotransmitter (Chesnoy-Marchais, 2009). These findings point to a potential mechanism by which ALLO may protect neuronal networks against excitotoxic damage in SZ (Plitman et al., 2014). The present finding suggests that low levels of ALLO may lead to reduced neuroprotection or result from an excessive consumption of ALLO engaged in neuroprotective action against excitotoxic damage observed in SZ.

4.3. Effect of antipsychotic treatment

In the present study, we demonstrated significantly lower levels of plasma ALLO in FEAN-SZ than in HC subjects (Table 1). However, in response to one month APD treatment, plasma levels of ALLO were markedly elevated (Fig. 2). Notably, the normalization of ALLO levels seen after 1 month of treatment, waned with continued treatment over the next 11 months. This may reflect compensatory changes with longer-term APD treatment and reduced efficacy with respect to effects on the neurosteroid pathways. Whether long-term APD treatment may be accompanied by tolerance over the longer run requires further investigation.

Given that 5α-reductase is the rate-limiting enzyme catalyzing the conversion from PROG to ALLO (Do Rego et al., 2009), the decrement in ALLO may reflect a parallel down-regulation of 5α-reductase. It is well known that some typical and atypical antipsychotic medications like haloperidol and risperidone could result in significant elevation of prolactin levels in SZ patient (David et al., 2000). Although APD-induced prolactin elevations can happen acutely, the serum prolactin levels will not peak until 10 weeks (Kinon et al., 2003). Therefore, this side effect may provide an alternate explanation for the reduced efficacy during the longer-term APD treatment since the APD-induced prolactin inhibits 5α-reductase expression (Serafini and Lobo, 1986).

In addition, ALLO is known to activate GABAA receptors, whose inhibitory action may indeed interact with potential changes in DA receptor signaling in dopaminergic regions (Motzo et al., 1996; Dazzi et al., 2002). Thus, certain atypical antipsychotics could potentially exert their therapeutic effects by increasing ALLO levels in the brain as well as in the blood. Consistent with this notion, there is now converging evidence that olanzapine is able to elevate ALLO to physiologically relevant concentrations in rodent cerebral cortex and hippocampus in a dose-dependent manner (Marx et al., 2000; Marx et al., 2006b; Marx et al., 2006c). Similar findings have also been obtained with clozapine, but not risperidone or haloperidol, in rats (Marx et al., 2006b; Barbaccia et al., 2001). Notably, Marx et al (2006b) have found that rat hippocampal PREG levels are strongly correlated with peripheral serum PREG levels, and it is possible that this association may also be present in humans.

Furthermore, Ugale et al (2004) indicated that administration of ALLO or the ALLO precursor, PROG significantly potentiated olanzapine-induced blockade of conditioned avoidance response and apomorphine-induced climbing in rodents. On the other hand, inhibition of the endogenous biosynthesis of neurosteroids or usage of GABAA receptor antagonist blocked the effect of olanzapine. These results suggest that ALLO-mediated GABAergic enhancement may contribute to the psychotropic-like action of olanzapine in rodents. Additionally, as stated earlier, preliminary data suggest the promise of ALLO as a therapeutic agent in SZ (Marx et al., 2009).

It is hypothesized that schizophrenia involves a dysregulation of dopaminergic neurotransmission in the brain with excess dopaminergic activity in the mesolimbic pathway as one of its essential manifestations (Miyamoto et al., 2012). Accumulating evidence indicates an inhibitory influence of GABAA receptors on the mesolimbic dopaminergic system (Xi and Stein, 1998). ALLO as a potent positive modulator of GABAA receptor is known to diminish basal and stress-induced dopaminergic transmission in the mesolimbic system by its action on GABAA receptors (Motzo et al, 1996). In the present study, lower basal levels of plasma ALLO with a decreased GABAergic tone may partially contribute to the hyperactivity of mesolimbic dopaminergic neurotransmission in FEAN-SZ. Previously, we have shown that plasma levels of GABA were significantly lower in SZ patients (11 FEAN-SZ and 21 relapsed antipsychotic-free SZ patients) than in controls (Cai et al., 2010), supporting the notion that GABAergic function is attenuated in SZ brain (Ohnuma et al., 2005). It is noteworthy that APDs do not exercise a direct action on the GABAA receptors (Schotte et al., 1996) but would assist in reversing an underactive GABAergic system in SZ (Farnbach-Pralong et al., 1998). Taken together, it is surmised that the APD-induced elevations in ALLO may increase the GABAergic tone which may, in turn, act to reduce the hyperdopaminergic tone, which may subsequently lead to an improvement in psychotic symptoms.

However, given another paradoxical report indicating that ALLO may increase the release of mesolimbic dopamine (Rouge-Pont et al., 2002), we have contemplated an opposite scenario that the observed reductions in basal ALLO levels of FEAN-SZ may be reflective of a homeostatic change to counteract the excessive mesolimbic dopaminergic activity in SZ. After the direct antagonism of mesolimbic dopamine receptors by APD during the first 4 weeks of treatment, medication-induced, reduction in mesolimbic dopaminergic tone would possibly trigger a compensating mechanism by an increment of ALLO levels. Thus, the possibility of ALLO and APD interaction with dopaminergic neurotransmission, in light of the indirect GABAergic modulation is still unclear at this stage and needs further elucidations.

4.4. Plasma neurosteroids and clinical relevancy

As proposed, we hypothesize that a homeostatic imbalance in the PREG-PROG-ALLO pathway exists early in the course of SZ. PROG is the precursor for the production of ALLO mediated by 5α-reductase and 3α-hydroxysteroid dehydrogenase reactions (Fig. 1). Lower levels of PROG will thus reflect a higher rate of metabolic conversion to ALLO. Although it is exploratory, it is not surprising to note that PROG level may serve as a predictor of medication response and therapeutic outcome. For example, plasma PROG levels at baseline were inversely correlated with GAS ratings at 1 month after APD (Fig. 3), suggesting that lower baseline PROG levels are associated with better functioning after APD treatment.

4.5. Limitations

Several limitations of our study should also be highlighted. Firstly, peripheral blood samples were used to study the deficits of PREG-PROG-ALLO pathway in FEAN-SZ. Do peripheral indices of metabolic deficits also reflect similar changes in the brain? This issue has been vigorously debated because of examples in the literature, where peripheral measures either failed to adequately reflect central pathophysiology or did not serve as reliable biological markers. In an editorial in Molecular Psychiatry, Wong and Licinio (2005) have eloquently stated that this belief has pervaded the field and has undermined our ability to confidently use the powerful tools of contemporary biology in order to dissect the biology of psychiatric disorders through investigation of peripheral markers, particularly those measured in peripheral blood. Previously, a good correlation between plasma and brain levels of PROG and ALLO has been demonstrated in rats (Barbaccia et al., 2001; Barbaccia et al., 1997). Moreover, Kim et al (2000) reported a high correlation of ALLO between plasma and cerebrospinal fluid in humans. A recent study also indicated that plasma levels of PREG and PROG were positively correlated with the corresponding indices in cerebral cortex in rats (Caruso et al., 2013). Thus, it is likely that our present findings showing peripheral alterations of the PREG-PROG-ALLO pathway may reflect similar changes in human brain regions.

Secondly, concerns have been raised about the adequacy of ELISA to measure steroid hormones in comparison with the state-of-the-art mass spectrometry (MS) method. Particularly, a recent study pointed out that the immunoassay methods can suffer from several shortcomings including susceptibility to sample matrix effects and lack of specificity due to antibody cross-reactivity (Broccardo et al., 2013). However, Dorgan et al (2002) pointed that ELISA and MS approaches could yield similar estimates of most targeted steroid hormones in human serum, although absolute concentrations may differ in some hormones. In the present study, we also compared normal ranges of our targeted neurosteroids measurements in serum/plasma between ELISA and MS assays (Supplementary Table 3). The commercial ELISA kits we used have acceptable cross-reactivity (the highest cross-reactivity 15% is between CORT and dexamethasone using CORT ELISA kit), high sensitivity, and low intra- and inter-assay variability (Supplementary Table 1). Furthermore, the normal range from our ELISA assay kits were also comparable to previously published data using MS method (Supplementary Table 3). Therefore, it is unlikely that our findings of significant differences between groups and across follow-up assessments are due to the methodological issues. Although we could not analyze these neurosteroids on both analytical platforms (MS and ELISA) due to economic considerations and sample availability, our findings will need to be further repeated in a larger cohort of FEAN-SZ patients by the LC-MS/MS measurements.

Thirdly, plasma levels of 5α-dihydroprogesterone (5α-DHP), the direct precursor of ALLO, were not measured due to the lack of a commercially available 5α-DHP ELISA kit. The data from an animal study have revealed that brain and plasma content of 5α-DHP and its precursor PROG would decrease markedly (70 and 80%, respectively) when adrenals and gonads were removed. To the contrary, ALLO content decreased only partially (30%) in the brain and remained low and virtually unchanged in plasma in spite of the fact that adrenalectomy and castration were performed at least 15 days before the determination (Cheney et al., 1995). It suggests that the association between brain and plasma ALLO levels may be mitigated from the peripheral influences as compared with 5α-DHP. Moreover, Ugale et al (2004) used indomethacin, a 3α-HSD enzyme inhibitor, to inhibit the conversion of 5α-DHP to ALLO in rats. Interestingly, the pretreatment with indomethacin which selectively reduced ALLO content without altering 5α-DHP levels abolished the behavioral actions of olanzapine. This result lends further support to the notion that metabolism of PROG to ALLO rather than to 5α-DHP is essential for the antipsychotic-like effect of olanzapine. Taken together, we hypothesize that ALLO, not 5α-DHP plays a pivotal role in the pathophysiology and treatment of schizophrenia.

5. Conclusion

In summary, we speculate that the PREG-PROG-ALLO pathway leading to ALLO production may represent a potential therapeutic target for APD development and treatment in SZ. It is also possible that 5α-reductase may play a pivotal role in the pathophysiology of SZ (Paba et al., 2011).

Supplementary Material

1
2
3

Highlights.

  • Plasma neurosteroids in pregnenolone (PREG) downstream pathways was compared between healthy controls (HC) and first-episode antipsychotic-naïve patients with schizophrenia (FEAN-SZ) before antipsychotic drug (APD) treatment.

  • Levels of PREG were significantly higher and those of allopregnanolone (ALLO) were lower in FEAN-SZ than in HC.

  • In response to APD for one month (1-m), ALLO levels in FEAN-SZ were markedly elevated whereas PREG and progesterone (PROG) levels decreased.

  • FEAN-SZ with lower levels of PROG (reflecting higher conversion to ALLO) at baseline may predict better therapeutic outcome after 1-m APD treatment.

  • The present findings point to the perturbations of PREG-PROG-ALLO pathway early in psychosis, and further study into this pathway may inform alternative and innovative therapeutic targets for SZ.

Acknowledgments

Role of the funding sources

This work was supported in part by Department of Veterans Affairs [Merit Reviews (JKY) and Senior Research Career Scientist Award (JKY)], the VA Pittsburgh Healthcare System, National Institute of Health [MH58141 (JKY), MH64023 (MK), KO2 MH 01180 (MK), MH45156 (David Lewis, MD, Director), NIH/NCRR/GCRC M01 RR00056 (Arthur Levine, MD)], Nature Science Foundation of China [NSFC81401113 (HLC)], the Specialized Research Fund for the Doctoral Program of Higher Education of China [SRFDP20130162120060 (HLC)], Hunan Provincial Natural Science Foundation of China [2017JJ3444 (HLC)], and the Chinese Scholarship Council for oversea study program (HLC).

The authors are grateful to Dr. Robert Gibbs and Mr. James Luther for their valuable comments. We thank the clinical core staff of the Center for the Neuroscience of Mental Disorders for their assistance in diagnostic and psychopathological assessments. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The contents of this article do not represent the views of the Department of Veterans Affairs, the United States Government, and its affiliated academic healthcare centers, or the National Institutes of Health.

Footnotes

Conflicts of interests

The authors declare no conflict of interest.

Contributors

HLC contributed to study design, data analysis and interpretation, drafting of the article, and final approval. GGD participated in statistical analysis and interpretation. XZ participated in laboratory assays. RDR, GLH, MK and DMM contributed to clinical design, subject recruitment, data interpretation, revising the article, and final approval. JKY contributed to the overall study design, laboratory assays, data analysis and interpretation, revising the article, and final approval.

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References

  1. Andreasen NC, Olsen S. Negative v positive schizophrenia. Definition and validation. Arch Gen Psychiatry. 1982;39:789–794. doi: 10.1001/archpsyc.1982.04290070025006. [DOI] [PubMed] [Google Scholar]
  2. Barbaccia ML, Affricano D, Purdy RH, Maciocco E, Spiga F, Biggio G. Clozapine, but not haloperidol, increases brain concentrations of neuroactive steroids in the rat. Neuropsychopharmacology. 2001;25:489–497. doi: 10.1016/S0893-133X(01)00254-8. [DOI] [PubMed] [Google Scholar]
  3. Barbaccia ML, Roscetti G, Trabucchi M, Purdy RH, Mostallino MC, Concas A, Biggio G. The effects of inhibitors of GABAergic transmission and stress on brain and plasma allopregnanolone concentrations. Br J harmacol. 1997;120:1582–1588. doi: 10.1038/sj.bjp.0701046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brambilla DJ, Matsumoto AM, Araujo AB, McKinlay JB. The effect of diurnal variation on clinical measurement of serum testosterone and other sex hormone levels in men. J Clin Endocrinol Metab. 2009;94:907–913. doi: 10.1210/jc.2008-1902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breier A, Buchanan RW. The effects of metabolic stress on plasma progesterone in healthy volunteers and schizophrenic patients. Life Sci. 1992;51:1527–1534. doi: 10.1016/0024-3205(92)90563-5. [DOI] [PubMed] [Google Scholar]
  6. Broccardo CJ, Schauer KL, Kohrt WM, Schwartz RS, Murphy JP, Prenni JE. Multiplexed analysis of steroid hormones in human serum using novel microflow tile technology and LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;934:16–21. doi: 10.1016/j.jchromb.2013.06.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cai HL, Zhu RH, Li HD, Zhang XH, Hu L, Yang W, Ye HS. Elevated plasma γ-aminobutyrate/glutamate ratio and responses to risperidone antipsychotic treatment in schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34:1273–1278. doi: 10.1016/j.pnpbp.2010.07.006. [DOI] [PubMed] [Google Scholar]
  8. Caruso D, Pesaresi M, Abbiati F, Calabrese D, Giatti S, Garcia-Segura LM, Melcangi RC. Comparison of plasma and cerebrospinal fluid levels of neuroactive steroids with their brain, spinal cord and peripheral nerve levels in male and female rats. Psychoneuroendocrinology. 2013;38:2278–2290. doi: 10.1016/j.psyneuen.2013.04.016. [DOI] [PubMed] [Google Scholar]
  9. Cheney DL, Uzunov D, Costa E, Guidotti A. Gas chromatographic-mass fragmentographic quantitation of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats. J Neurosci. 1995;15:4641–4650. doi: 10.1523/JNEUROSCI.15-06-04641.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chesnoy-Marchais D. Progesterone and allopregnanolone enhance the miniature synaptic release of glycine in the rat hypoglossal nucleus. Eur J Neurosci. 2009;30:2100–2111. doi: 10.1111/j.1460-9568.2009.07013.x. [DOI] [PubMed] [Google Scholar]
  11. Condray R, Dougherty GG, Jr, Keshavan MS, Reddy RD, Haas GL, Montrose DM, Matson WR, McEvoy J, Kaddurah-Daouk R, Yao JK. 3-Hydroxykynurenine and clinical symptoms in first-episode neuroleptic-naïve patients with schizophrenia. Int J Neuropsychopharmacol. 2011;14:756–767. doi: 10.1017/S1461145710001689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. David SR, Taylor CC, Kinon BJ, Breier A. The effects of olanzapine, risperidone, and haloperidol on plasma prolactin levels in patients with schizophrenia. Clin Ther. 2000;22:1085–1096. doi: 10.1016/S0149-2918(00)80086-7. [DOI] [PubMed] [Google Scholar]
  13. Dazzi L, Serra M, Vacca G, Ladu S, Latrofa A, Trapani G, Biggio G. Depletion of cortical allopregnanolone potentiates stress-induced increase in cortical dopamine output. Brain Res. 2002;932:135–139. doi: 10.1016/s0006-8993(02)02290-4. [DOI] [PubMed] [Google Scholar]
  14. Djebaili M, Hoffman SW, Stein DG. Allopregnanolone and progesterone decrease cell death and cognitive deficits after a contusion of the rat pre-frontal cortex. Neuroscience. 2004;123:349–359. doi: 10.1016/j.neuroscience.2003.09.023. [DOI] [PubMed] [Google Scholar]
  15. Djebaili M, Guo Q, Pettus EH, Hoffman SW, Stein DG. The neurosteroids progesterone and allopregnanolone reduce cell death, gliosis, and functional deficits after traumatic brain injury in rats. J Neurotrauma. 2005;22:106–118. doi: 10.1089/neu.2005.22.106. [DOI] [PubMed] [Google Scholar]
  16. Do Rego JL, Seong JY, Burel D, Leprince J, Luu-The V, Tsutsui K, Tonon MC, Pelletier G, Vaudry H. Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front Neuroendocrinol. 2009;30:259–301. doi: 10.1016/j.yfrne.2009.05.006. [DOI] [PubMed] [Google Scholar]
  17. Dorgan JF, Fears TR, McMahon RP, Aronson Friedman L, Patterson BH, Greenhut SF. Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry. Steroids. 2002;67:151–158. doi: 10.1016/s0039-128x(01)00147-7. [DOI] [PubMed] [Google Scholar]
  18. Endicott J, Spitzer RL, Fleiss JL, Cohen J. The global assessment scale. A procedure for measuring overall severity of psychiatric disturbance. Arch Gen Psychiatry. 1976;33:766–771. doi: 10.1001/archpsyc.1976.01770060086012. [DOI] [PubMed] [Google Scholar]
  19. Farnbach-Pralong D, Bradbury R, Copolov D, Dean B. Clozapine and olanzapine treatment decreases rat cortical and limbic GABA(A) receptors. Eur J Pharmacol. 1998;349:R7–8. doi: 10.1016/s0014-2999(98)00285-4. [DOI] [PubMed] [Google Scholar]
  20. Guo Q, Sayeed I, Baronne LM, Hoffman SW, Guennoun R, Stein DG. Progesterone administration modulates AQP4 expression and edema after traumatic brain injury in male rats. Exp Neurol. 2006;198:469–478. doi: 10.1016/j.expneurol.2005.12.013. [DOI] [PubMed] [Google Scholar]
  21. Guy W, editor. ECDEU Assessment Manual for Psychopharmacology, revised. National Institute of Mental Health; Rockville, MD: 1976. Clinical Global Impressions. [Google Scholar]
  22. Hatzimanikatis V, Li C, Ionita JA, Broadbelt LJ. Metabolic networks: enzyme function and metabolite structure. Curr Opin Struct Biol. 2004;14:300–306. doi: 10.1016/j.sbi.2004.04.004. [DOI] [PubMed] [Google Scholar]
  23. He J, Evans CO, Hoffman SW, Oyesiku NM, Stein DG. Progesterone and allopregnanolone reduce inflammatory cytokines after traumatic brain injury. Exp Neurol. 2004;189:404–412. doi: 10.1016/j.expneurol.2004.06.008. [DOI] [PubMed] [Google Scholar]
  24. Jaworska-Feil L, Budziszewska B, Leśkiewicz M, Lasoń W. Opposite effects of inhibitory and excitatory neurosteroids on [3H]dopamine release from rat nucleus accumbens. Pol J Pharmacol. 1998;50:449–452. [PubMed] [Google Scholar]
  25. Johnson RA, Wichern DW. Applied Multivariate Statistical Analysis. 4. Upper Saddle River, NJ: Prentice Hall; 1998. pp. 193–194. [Google Scholar]
  26. Khisti RT, Deshpande LS, Chopde CT. The neurosteroid 3α-hydroxy-5α-pregnan-20-one affects dopamine-mediated behavior in rodents. Psychopharmacology (Berl) 2002;161:120–128. doi: 10.1007/s00213-002-1006-5. [DOI] [PubMed] [Google Scholar]
  27. Kim YS, Zhang H, Kim HY. Profiling neurosteroids in cerebrospinal fluids and plasma by gas chromatography/electron capture negative chemical ionization mass spectrometry. Anal Biochem. 2000;277:187–195. doi: 10.1006/abio.1999.4384. [DOI] [PubMed] [Google Scholar]
  28. Kinon BJ, Gilmore JA, Liu H, Halbreich UM. Hyperprolactinemia in response to antipsychotic drugs: characterization across comparative clinical trials. Psychoneuroendocrinology. 2003;28:69–82. doi: 10.1016/s0306-4530(02)00128-2. [DOI] [PubMed] [Google Scholar]
  29. Lan NC, Gee KW. Neuroactive steroid actions at the GABAA receptor. Horm Behav. 1994;28:537–544. doi: 10.1006/hbeh.1994.1052. [DOI] [PubMed] [Google Scholar]
  30. Lehmann EL. Nonparametrics: Statistical Methods Based on Ranks. San Francisco: Holden and Day; 1975. [Google Scholar]
  31. Lewis DA, Volk DW, Hashimoto T. Selective alterations in prefrontal cortical GABA neurotransmission in schizophrenia: a novel target for the treatment of working memory dysfunction. Psychopharmacology. 2004;74:143–150. doi: 10.1007/s00213-003-1673-x. [DOI] [PubMed] [Google Scholar]
  32. MacKenzie EM, Odontiadis J, Le Mellédo JM, Prior TI, Baker GB. The relevance of neuroactive steroids in schizophrenia, depression, and anxiety disorders. Cell Mol Neurobiol. 2007;27:541–574. doi: 10.1007/s10571-006-9086-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marx CE, Bradford DW, Hamer RM, Naylor JC, Allen TB, Lieberman JA, Strauss JL, Kilts JD. Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and clinical evidence. Neuroscience. 2011;191:78–90. doi: 10.1016/j.neuroscience.2011.06.076. [DOI] [PubMed] [Google Scholar]
  34. Marx CE, Duncan GE, Gilmore JH, Lieberman JA, Morrow AL. Olanzapine increases allopregnanolone in the rat cerebral cortex. Biol Psychiatry. 2000;47:1000–1004. doi: 10.1016/s0006-3223(99)00305-4. [DOI] [PubMed] [Google Scholar]
  35. Marx CE, Keefe RS, Buchanan RW, Hamer RM, Kilts JD, Bradford DW, Strauss JL, Naylor JC, Payne VM, Lieberman JA, Savitz AJ, Leimone LA, Dunn L, Porcu P, Morrow AL, Shampine LJ. Proof-of-concept trial with the neurosteroid pregnenolone targeting cognitive and negative symptoms in schizophrenia. Neuropsychopharmacology. 2009;34:1885–1903. doi: 10.1038/npp.2009.26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marx CE, Lee J, Subramaniam M, Rapisarda A, Bautista DC, Chan E, Kilts JD, Buchanan RW, Wai EP, Verma S, Sim K, Hariram J, Jacob R, Keefe RS, Chong SA. Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia. Psychopharmacology (Berl) 2014;231:3647–3662. doi: 10.1007/s00213-014-3673-4. [DOI] [PubMed] [Google Scholar]
  37. Marx CE, Shampine LJ, Duncan GE, VanDoren MJ, Grobin AC, Massing MW, Madison RD, Bradford DW, Butterfield MI, Lieberman JA, Morrow AL. Clozapine markedly elevates pregnenolone in rat hippocampus, cerebral cortex, and serum: candidate mechanism for superior efficacy? Pharmacol Biochem Behav. 2006b;84:598–608. doi: 10.1016/j.pbb.2006.07.026. [DOI] [PubMed] [Google Scholar]
  38. Marx CE, Shampine LJ, Khisti RT, Trost WT, Bradford DW, Grobin AC, Massing MW, Madison RD, Butterfield MI, Lieberman JA, Morrow AL. Olanzapine and fluoxetine administration and coadministration increase rat hippocampal pregnenolone, allopregnanolone and peripheral deoxycorticosterone: implications for therapeutic actions. Pharmacol Biochem Behav. 2006c;84:609–617. doi: 10.1016/j.pbb.2006.07.032. [DOI] [PubMed] [Google Scholar]
  39. Marx CE, Stevens RD, Shampine LJ, Uzunova V, Trost WT, Butterfield MI, Massing MW, Hamer RM, Morrow AL, Lieberman JA. Neuroactive steroids are altered in schizophrenia and bipolar disorder: relevance to pathophysiology and therapeutics. Neuropsychopharmacology. 2006a;31:1249–1263. doi: 10.1038/sj.npp.1300952. [DOI] [PubMed] [Google Scholar]
  40. Mensah-Nyagan AG, Do-Rego JL, Beaujean D, Luu-The V, Pelletier G, Vaudry H. Neurosteroids: expression of steroidogenic enzymes and regulation of steroid biosynthesis in the central nervous system. Pharmacol Rev. 1999;51:63–81. [PubMed] [Google Scholar]
  41. Miyamoto S, Miyake N, Jarskog LF, Fleischhacker WW, Lieberman JA. Pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents. Mol Psychiatry. 2012;17:1206–1227. doi: 10.1038/mp.2012.47. [DOI] [PubMed] [Google Scholar]
  42. Motzo C, Porceddu ML, Maira G, Flore G, Concas A, Dazzi L, Biggio G. Inhibition of basal and stress-induced dopamine release in the cerebral cortex and nucleus accumbens of freely moving rats by the neurosteroid allopregnanolone. J Psychopharmacol. 1996;10:266–272. doi: 10.1177/026988119601000402. [DOI] [PubMed] [Google Scholar]
  43. Ohnuma T, Suzuki T, Arai H. Hypothesis: minimal changes in neural transmission in schizophrenia: decreased glutamatergic and GABAergic functions in the prefrontal cortex. Prog Neuropsychopharmacol Biol Psychiatry. 2005;29:889–894. doi: 10.1016/j.pnpbp.2005.04.038. [DOI] [PubMed] [Google Scholar]
  44. Overall JE, Gorham DR. The Brief Psychiatric Rating Scale. Psychol Rep. 1962;10:779–812. [Google Scholar]
  45. Paba S, Frau R, Godar SC, Devoto P, Marrosu F, Bortolato M. Steroid 5α-reductase as a novel therapeutic target for schizophrenia and other neuropsychiatric disorders. Curr Pharm Des. 2011;17:151–167. doi: 10.2174/138161211795049589. [DOI] [PubMed] [Google Scholar]
  46. Plitman E, Nakajima S, de la Fuente-Sandoval C, Gerretsen P, Chakravarty MM, Kobylianskii J, Chung JK, Caravaggio F, Iwata Y, Remington G, Graff-Guerrero A. Glutamate-mediated excitotoxicity in schizophrenia: a review. Eur Neuropsychopharmacol. 2014;24:1591–1605. doi: 10.1016/j.euroneuro.2014.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ritsner M, Maayan R, Gibel A, Weizman A. Differences in blood pregnenolone and dehydroepiandrosterone levels between schizophrenia patients and healthy subjects. Eur Neuropsychopharmacol. 2007;17:358–365. doi: 10.1016/j.euroneuro.2006.10.001. [DOI] [PubMed] [Google Scholar]
  48. Robertson CL, Puskar A, Hoffman GE, Murphy AZ, Saraswati M, Fiskum G. Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats. Exp Neurol. 2006;197:235–243. doi: 10.1016/j.expneurol.2005.09.014. [DOI] [PubMed] [Google Scholar]
  49. Rouge-Pont F, Mayo W, Marinelli M, Gingras M, Le Moal M, Piazza PV. The neurosteroid allopregnanolone increases dopamine release and dopaminergic response to morphine in the rat nucleus accumbens. Eur J Neurosci. 2002;16:169–173. doi: 10.1046/j.1460-9568.2002.02084.x. [DOI] [PubMed] [Google Scholar]
  50. Sayeed I, Parvez S, Wali B, Siemen D, Stein DG. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism for better neuroprotective effects of allopregnanolone over progesterone. Brain Res. 2009;1263:165–173. doi: 10.1016/j.brainres.2009.01.045. [DOI] [PubMed] [Google Scholar]
  51. Schotte A, Janssen PF, Gommeren W, Luyten WH, Van Gompel P, Lesage AS, De Loore K, Leysen JE. Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology (Berl) 1996;124:57–73. doi: 10.1007/BF02245606. [DOI] [PubMed] [Google Scholar]
  52. Serafini P, Lobo RA. Prolactin modulates peripheral androgen metabolism. Fertil Steril. 1986;45:41–46. [PubMed] [Google Scholar]
  53. Shirayama Y, Hashimoto K, Suzuki Y, Higuchi T. Correlation of plasma neurosteroid levels to the severity of negative symptoms in male patients with schizophrenia. Schizophr Res. 2002;58:69–74. doi: 10.1016/s0920-9964(01)00367-x. [DOI] [PubMed] [Google Scholar]
  54. Skolnick BE, Maas AI, Narayan RK, van der Hoop RG, MacAllister T, Ward JD, Nelson NR, Stocchetti N. A clinical trial of progesterone for severe traumatic brain injury. N Engl J Med. 2014;371:2467–2476. doi: 10.1056/NEJMoa1411090. [DOI] [PubMed] [Google Scholar]
  55. Ugale RR, Hirani K, Morelli M, Chopde CT. Role of neuroactive steroid allopregnanolone in antipsychotic-like action of olanzapine in rodents. Neuropsychopharmacology. 2004;29:1597–1609. doi: 10.1038/sj.npp.1300460. [DOI] [PubMed] [Google Scholar]
  56. VanLandingham JW, Cekic M, Cutler SM, Hoffman SW, Washington ER, Johnson SJ, Miller D, Stein DG. Progesterone and its metabolite allopregnanolone differentially regulate hemostatic proteins after traumatic brain injury. J Cereb Blood Flow Metab. 2008;28:1786–1794. doi: 10.1038/jcbfm.2008.73. [DOI] [PubMed] [Google Scholar]
  57. Verma SK, Subramaniam M, Liew A, Poon LY. Metabolic risk factors in drug-naive patients with first-episode psychosis. J Clin Psychiatry. 2009;70:997–1000. doi: 10.4088/JCP.08m04508. [DOI] [PubMed] [Google Scholar]
  58. Wadenberg ML, Soliman A, VanderSpek SC, Kapur S. Dopamine D(2) receptor occupancy is a common mechanism underlying animal models of antipsychotics and their clinical effects. Neuropsychopharmacology. 2001;25:633–641. doi: 10.1016/S0893-133X(01)00261-5. [DOI] [PubMed] [Google Scholar]
  59. Welch BL. The generalisation of student’s problems when several different population variances are involved. Biometrika. 1947;34:28–35. doi: 10.1093/biomet/34.1-2.28. [DOI] [PubMed] [Google Scholar]
  60. Williams JB. A structured interview guide for the Hamilton Depression Rating Scale. Arch Gen Psychiatry. 1988;45:742–747. doi: 10.1001/archpsyc.1988.01800320058007. [DOI] [PubMed] [Google Scholar]
  61. Wong ML, Licinio J. Peripheral is central to the question. Mol Psychiatry. 2005;10:421–422. doi: 10.1038/sj.mp.4001674. [DOI] [PubMed] [Google Scholar]
  62. Xi ZX, Stein EA. Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors-an in vivo electrochemical study. Brain Res. 1998;798:156–165. doi: 10.1016/s0006-8993(98)00406-5. [DOI] [PubMed] [Google Scholar]
  63. Zhang Y, Xiong Y, Mahmood A, Meng Y, Liu Z, Qu C, Chopp M. Sprouting of corticospinal tract axons from the contralateral hemisphere into the denervated side of the spinal cord is associated with functional recovery in adult rat after traumatic brain injury and erythropoietin treatment. Brain Res. 2010;1353:249–257. doi: 10.1016/j.brainres.2010.07.046. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

1
NIHMS941916-supplement-1.doc (28KB, doc)
2
NIHMS941916-supplement-2.doc (47.5KB, doc)
3
NIHMS941916-supplement-3.doc (30.5KB, doc)

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