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GABAA receptor active steroids are altered in epilepsy patients with tuberous sclerosis
  1. F di Michele1,
  2. M Verdecchia2,
  3. M Dorofeeva4,
  4. L Costamagna3,
  5. G Bernardi2,
  6. P Curatolo2,
  7. E Romeo1
  1. 1Neuroendocrinology Laboratory, IRCCS Santa Lucia, Rome, Italy
  2. 2Department of Neuroscience, Tor Vergata University, Rome
  3. 3Department of Forensic Medicine, La Sapienza University, Rome
  4. 4Department of Paediatric Neurology, University of Moscow, Moscow, Russia
  1. Correspondence to:
 Dr Flavia di Michele, IRCCS Santa Lucia, via Ardeatina 306, 00179 Rome, Italy; 
 f.dimichele{at}hsantalucia.it

Abstract

Background: The neuroactive steroid 3α, 5α-tetrahydroprogesterone is the most potent endogenous positive modulator of γ-amino-butyric acid (GABA)A receptors. There is evidence for a relation between neuroactive steroids and seizure susceptibility.

Objective: To evaluate the putative role of counteregulator neuroactive steroids in the occurrence of seizures in patients with tuberous sclerosis.

Methods: Plasma concentrations of the enantiomers 3α, 5α- and 3α, 5β-tetrahydroprogesterone (3αs-THP), which are positive modulators of GABAA receptors, were measured in 18 patients, along with their endogenous functional antagonists 3β, 5α- and 3β, 5β-THP (3βs-THP), to assess their possible modification compared with control subjects. Neuroactive steroids were assayed using a highly sensitive and specific gas chromatographic/mass spectrometric method.

Results: In the tuberous sclerosis patients with poorly controlled seizures, there was a significantly lower 3αs/3βs-THP ratio than in seizure-free patients or control subjects.

Conclusions: The reduced 3αs/3βs-THP ratio may decrease GABAergic tone, contributing to the appearance of seizures in tuberous sclerosis patients with epilepsy.

  • tetrahydroprogesterone
  • epilepsy
  • neuroactive steroids
  • tuberous sclerosis
  • GABA
  • γ-amino-butyric acid
  • s-THP, 3α, 5α- and 3α, 5β-tetrahydroprogesterone
  • s-THP
  • 5α- and 3β
  • 5β-THP (functional antagonists of 3αs-THP

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Tuberous sclerosis is a disorder characterised by hamartomas that can affect any organ. In the brain, cortical tubers are regions of disorganised cortical lamination and are directly related to the presence of partial seizures that are often resistant to medical treatment.1

Surprisingly, a large increase in γ-amino-butyric acid (GABA) has been reported in brain biopsies of subjects affected by tuberous sclerosis who have intractable epilepsy, compared with intractable epilepsy from other causes.2 Moreover, vigabatrin, a specific and irreversible inhibitor of GABA-aminotransferase, is more effective at treating infantile spasms and partial seizures caused by tuberous sclerosis than other types of epilepsy.3,4 These findings suggest the fundamental involvement of GABAergic neurotransmission in the epileptogenesis of tuberous sclerosis. However, rather than being caused by a defect in GABA biosynthesis, the pathogenesis of epilepsy in tuberous sclerosis could involve an alteration of the GABAA receptor subunit assembly and structure,5 or a modified expression of endogenous GABAA receptor modulatory agents. The neuroactive steroids 3α, 5α- and 3α, 5β-tetrahydroprogesterone (3αs-THP) are potent positive allosteric modulators of GABAA receptors.6,7 Altered 3αs-THP concentrations could be one of the reasons for the increased brain GABA levels in patients with tuberous sclerosis.2 The rationale for this hypothesis is based on the following:

  • Pharmacological studies have shown that 3αs-THP-reduced metabolites of progesterone are positive GABAA receptor modulators, while the 3βs-THP enantiomers (3β, 5α-THP and 3β, 5β-THP) reduce the ability of 3αs-THP to potentiate GABAA receptor function, acting as functional antagonists.8–11 Moreover, antiepileptic effects of 3αs-THP have been demonstrated in humans and in animal models with partial epilepsy and well defined epileptic foci.12,13 In addition, 3αs-THP may be effective only against convulsive seizures, including epilepsy caused by tuberous sclerosis, but not against non-convulsive seizures.14

  • There is biochemical evidence from human studies showing that 3αs-THP and its stereoisomers are synthesised in the brain and in the periphery from progesterone.15

  • Behavioural and electrophysiological studies have shown that a decrease in brain 3αs-THP results in a decreased GABAA receptor efficacy and in a reduction of the hypnotic/anaesthetic action of several GABAmimetic drugs.16

These data strongly suggest that 3αs-THP plays an important permissive physiological role in the action of GABA at GABAA receptors. We therefore assayed 3αs-THP, 3βs-THP, and progesterone concentrations with the appropriate gas chromatographic/mass spectrometric (GC/MS) technique,17,18 anticipating that an alteration in their plasma levels might reflect a genetic or epigenetic defect in the enzymatic machinery that regulates THP synthesis.

METHODS

We studied 18 patients with tuberous sclerosis and a history of epilepsy who were referred to the department of neurology of Moscow University. As shown in table 1, of these 18 patients, six had been seizure-free for at least 2.5 years (patients 1 to 6), four were experiencing monthly seizures (patients 7 to 10), four had weekly seizures (patients 11 to 14), and four had daily seizures (patients 15 to 18). Clinical information on the patients is summarised in table 1. The diagnosis of tuberous sclerosis had been made using the new revised clinical diagnostic criteria.19

Table 1

Clinical profile of the patients with tuberous sclerosis

Six healthy subjects matched for age and sex referred to the clinic for a previous infective disease formed the control group (three male, three female; mean (SD) age 10.8 (10.8) years).

Exclusion criteria were as follows: neurological disorders other than tuberous sclerosis; endocrinological and dismetabolic disorders; and other pharmacological treatments in the previous three months.

The study was approved by the local ethics committee, and informed consent was obtained from the patients or their parents before investigations were carried out.

Heparinised blood samples were taken between 9 am and 10 am during the interictal period for plasma determination of 3αs-THP, 3βs-THP, and progesterone. In patients 7 to 18, carbamazepine and valproic acid were given 12 to 13 hours before blood sampling. In female subjects with menstrual cycles, the blood samples were taken during the follicular phase.

We used a GC-MS method for the determination of neuroactive steroids.17,18 This profiles and quantifies very small amounts (fmol) of steroidal compounds with high accuracy and allowed us to identify the stereoisomers. Briefly, approximately 5000 dpm of [H3]-progesterone were added to the plasma to monitor recovery. Ethyl acetate was used for extraction (3 × 2 ml) and thin layer chromatography for separation (2 mm silica gel 60; solvent systems: carbon tetrachloride/methanol 99:1 (vol/vol), followed by a consecutive run in one direction using cyclohexane/ethyl acetate (3:2, vol/vol)). We then added 7 pmol of progesterone to the eluate containing 3αs-THP or 3βs-THP as an internal standard, and 10 pmol of 3α,α-THP to the eluate containing progesterone. These eluates were freeze dried and derivatised with heptafluorobutyric acid anhydride. Derivatised steroids were analysed using a Finnigan Trace GC/MS instrument equipped with a capillary column (HP-35MS: length 30 m, internal diameter 0.25 mm, film thickness 0.25 μm). The derivatised steroids were assayed in the negative ion chemical ionisation mode. The recovery of tritiated steroids ranged from 80% to 90%. The detection limit for the steroids studied was approximately 10 fmol.

Statistical analysis

Differences in steroid concentrations and in the ratio between 3αs-THP and 3βs-THP within the tuberous sclerosis groups of patients and the control subjects were tested for significance using a one factor analysis of variance (ANOVA). The Tukey–Kramer test was subsequently used for any steroid or 3αs/3βs-THP ratio that showed a significant effect at p < 0.05 on ANOVA. Statistical analysis was done using SPSS for Windows 10.8.

RESULTS

No significant age effect was detected by ANOVA between the groups of tuberous sclerosis patients and controls. No significant differences were found between the tuberous sclerosis groups for age of seizure onset and the number or localisation of cortical tubers.

In comparisons of steroid concentrations, ANOVA showed significant differences in the concentrations of 3αs-THP and 3βs-THP between seizure-free tuberous sclerosis patients, tuberous sclerosis patients with epilepsy, and the control group (fig 1), but not in the concentrations of progesterone (F = 2.36; df = 159.54; p = 0.08).

Figure 1

Histograms showing: (A) mean and SD (error bars) of the neuroactive steroids 3αs-THP and 3βs-THP; (B) Mean and SD of the ratio between 3αs-THP and 3βs-THP values, in control subjects and patients with tuberous sclerosis (TS) seizure-free, or with monthly, weekly, and daily seizure frequencies. Analysis of variance probability (p) values are shown above each group of neuroactive steroids and their ratio; asterisks and daggers indicate significant post hoc results. (A) Post hoc results showed that 3αs-THP levels were decreased in TS patients with daily seizures compared with control subjects (p < 0.05) and seizure-free TS patients (p < 0.05). The 3βs-THP concentrations were increased in TS patients with monthly, weekly, and daily seizures compared with control subjects (p < 0.0001, p < 0.0001, and p < 0.005, respectively) and seizure-free TS patients (p < 0.0001, p < 0.0001, and p < 0.005, respectively). (B) Post hoc results showed that the ratio between 3αs-THP and 3βs-THP values was decreased in TS patients with monthly, weekly, and daily seizures compared with control subjects (p < 0.0001, p < 0.0001, and p < 0.0001, respectively) and seizure-free TS patients (p < 0.0001, p < 0.0001, and p < 0.0001, respectively). *Post hoc significance compared with control subjects; †Post hoc significance compared with seizure-free TSC patients. 3αs-THP, 3α, 5α-tetrahydroprogesterone and 3α, 5β-tetrahydroprogesterone; 3βs-THP, 3β, 5α-tetrahydroprogesterone and 3β, 5β-tetrahydroprogesterone.

As shown in fig 1, plasma levels of 3βs-THP were increased in all tuberous sclerosis patients with epilepsy, independent of seizure frequency, when compared with seizure-free patients and control subjects. The concentrations of 3αs-THP were unchanged in patients with monthly seizures, were decreased (although not significantly) in patients with weekly seizures, and were significantly reduced in patients with daily seizures compared with controls and with seizure-free tuberous patients.

There was a highly significant decrease in the ratio 3αs/3βs-THP in all tuberous sclerosis patients with epilepsy compared with seizure-free patients and controls (fig 1).

DISCUSSION

The 3αs-THP metabolites are potent positive allosteric modulators of GABA action at GABAA receptors in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In contrast, the 3βs-THP are devoid of intrinsic activity at GABAA receptors and behave as functional antagonists of the actions of 3αs-THP in potentiating GABAA receptor function.8–11

Various investigators have shown that naturally occurring 3α reduced neuroactive steroids (such as 3αs-THP) exert anticonvulsant effects.12,13 To our knowledge, no similar investigations have examined the 3β reduced compounds, owing to the difficulty in detecting stereoisomers by simple radioimmunoassay methods. Thus this is the first clinical study on epilepsy that has evaluated the entire spectrum of THP enantiomers by means of a GC-MS technique, which is highly sensitive and specific.18,19

The finding of unchanged THP enantiomer plasma levels in seizure-free tuberous sclerosis patients compared with controls may suggest that alterations in neuroactive steroids are not primary in tuberous sclerosis but represent an epiphenomenon related to seizures or their pharmacological treatment.

The reduced 3αs-THP concentrations found only in patients with daily seizures, in which the interictal period is very short, may be caused by seizure induced stress. However, in a previous study by Galli et al it was reported that there was an increase in allopregnanolone (3α, 5α-THP) in the postictal phase.13 A possible explanation for this discrepancy is that the radioimmunoassay method used in that study did not discriminate between the different enantiomers and thus considered the whole pool of THP stereoisomers. Furthermore, they reported increased allopregnanolone levels up to 15 minutes after seizure occurrence, which is a very short time span compared with our paradigm, where the minimum interval was never less than one hour.

However, the major finding of our study was the decreased ratio between 3αs and 3βs-THP in all tuberous sclerosis patients with epilepsy (fig 1B). This ratio should be considered the most relevant index of GABAA receptor function. A decrease in the modulatory effect of 3αs-THP, caused mainly by the augmented 3βs-THP levels in the brain, could result in a reduced sensitivity of GABAA receptors to GABA, and in a great reduction in the sedative anticonvulsant action of several GABAmimetic drugs.16 Thus the high GABA levels found in brain biopsies of drug resistant tuberous sclerosis patients2 could represent a compensatory mechanism for the downregulation of GABAA receptor function resulting from a decreased 3αs/3βs-THP ratio.

These findings are consistent with the evidence of a relation between neuroactive steroids and seizure susceptibility.12 A possible hypothesis to explain our data is that there might be an inadequate induction of the enzymes 5α/5β-reductases or the 3α/3β-hydroxysteroid oxidoreductases responsible for the biosynthesis of the 3αs or 3βs reduced derivatives of progesterone in tuberous sclerosis patients with seizures.15,20

On the other hand, treatment with antiepileptic drugs could cause changes in the enzymes that metabolise 3αs- or 3βs-THP. It has been shown, for instance, that carbamazepine induces steroidogenesis in rat brain, increasing 3α, 5α-THP concentrations.21 However, we did not found significant changes in the 3αs-THP levels except for a decrement in patients with daily seizures.

Although we need further analyses of samples from a larger population of patients with tuberous sclerosis before and after antiepileptic treatment, and also appropriate pharmacological studies in animal models, the decreased 3αs/3βs-THP ratio in the patients with epilepsy may reflect an altered GABAA receptor mediated excitability which could facilitate the occurrence of seizures.

Conclusions

This preliminary evidence is significant not only for a better understanding of the pathophysiology of seizures but also in the clinical perspective in tuberous sclerosis, as it opens the field to the development of possible therapeutic agents. Recently, a 3αs-THP analogue, ganaxolone, with anticonvulsant properties in various seizure models such as partial epilepsy, has been synthesised.22 Ganaxolone might also be considered as a potential therapeutic agent in patients with tuberous sclerosis who have epilepsy.

Acknowledgments

We would like to thank C Peter Bengtson for help with the English language.

REFERENCES

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Footnotes

  • Competing interests: none declared

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