Andrew Makoff

Do antidepressants regulate how cortisol affects the brain

Although the effects of antidepressants on glucocorticoid hormones and their receptors are relevant for the therapeutic action of these drugs, the molecular mechanisms underlying these effects are unclear. Studies in depressed patients, animals and cellular models have demonstrated that antidepressants increase glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) expression and function; this, in turn, is associated with enhanced negative feedback by endogenous glucocorticoids, and thus with reduced resting and stimulated hypothalamic-pituitary-adrenal (HPA) axis activity. In a series of studies conducted over the last few years, we have shown that antidepressants modulate GR function in vitro by inhibiting membrane steroid transporters that regulate the intracellular concentration of glucocorticoids. In this paper, we will review the effects of membrane steroid transporters and antidepressants on corticosteroid receptors. We will then present our unpublished data on GR live microscopy in vitro, showing that ligand-induced translocation of the GR starts within 30 seconds and is completed within minutes. Furthermore, we will present our new data using an in situ brain perfusion model in anaesthetised guinea-pigs, showing that entry of cortisol to the brain of these animals is limited at the blood-brain barrier (BBB). Finally, we will present a comprehensive discussion of our published findings on the effects of chemically unrelated antidepressants on membrane steroid transporters, in mouse fibroblasts and rat cortical neurones. We propose that antidepressants in humans could inhibit steroid transporters localised on the BBB and in neurones, like the multidrug resistance p-glycoprotein, and thus increase the access of cortisol to the brain and the glucocorticoid-mediated negative feedback on the HPA axis. Enhanced cortisol action in the brain might prove to be a successful approach to maximise therapeutic antidepressant effects.

Risk Factors in Sudden Death in Epilepsy (SUDEP): The Quest for Mechanisms

Summary:  People with epilepsy may die suddenly and unexpectedly without a structural pathological cause. Most SUDEP cases are likely to be related to seizures. SUDEP incidence varies and is <1:1,000 person‐years among prevalent cases in the community and ∼1:250 person years in specialist centres. Case–control studies identified certain risk factors, some potentially amenable to manipulation, including uncontrolled convulsive seizures and factors relating to treatment and supervision. Both respiratory and cardiac mechanisms are important. The apparent protective effect of lay supervision supports an important role for respiratory factors, in part amenable to intervention by simple measures. Whereas malignant tachyarrhythmias are rare during seizures, sinus bradycardia/arrest, although infrequent, is well documented. Both types of arrhythmias can have a genetic basis. This article reviews SUDEP and explores the potential of coexisting liability to cardiac arrhythmias as a contributory factor, while acknowledging that at present, bridging evidence between cardiac inherited gene determinants and SUDEP is lacking.

RNA editing of the 5-HT(2C) receptor is reduced in schizophrenia.

5-HT2C receptor (5HT2CR, serotonin-2C) RNA undergoes editing to produce several receptor variants, some with pharmacological differences. This investigation comprised two parts: the characterisation of 5-HT2CR RNA editing in a larger human control sample than previously examined, and a comparative study in subjects with schizophrenia. Secondary structure analysis of the putative edited region of the human 5-HT2CR gene predicted the existence of a double stranded (ds) RNA loop, essential for RNA editing in this receptor. RNA was then extracted from frontal cortex of five controls and five subjects with schizophrenia. RT-PCR products of the edited region were cloned and sequenced (n = 100). Reduced RNA editing, increased expression of the unedited 5-HT2C-INI isoform in schizophrenia (P = 0.001) and decreased expression of the 5-HT2C-VSV and 5-HT2C-VNV isoforms were detected in the schizophrenia group. In addition, two novel mRNA edited variants were identified: 5-HT2C-MNI and 5-HT2C-VDI. Screening of the 5-HT2CR gene did not reveal any mutations likely to disrupt the dsRNA loop, suggesting that the reduced RNA editing in schizophrenia may instead be caused by altered activity of the editing enzyme(s). Since the unedited 5-HT2C-INI is more efficiently coupled to G proteins than the other isoforms, its increased expression in schizophrenia may lead to enhanced 5-HT2CR-mediated effects. The results also illustrate that potentially important receptor alterations may occur in schizophrenia which are not detectable merely in terms of receptor abundance.

Antidepressants enhance glucocorticoid receptor function in vitro by modulating the membrane steroid transporters.

Previous data demonstrate that the tricyclic antidepressant, desipramine, induces glucocorticoid receptor (GR) translocation from the cytoplasm to the nucleus in L929 cells and increases dexamethasone‐induced GR‐mediated gene transcription in L929 cells stably transfected with the mouse mammary tumour virus‐chloramphenicol acetyltransferase (MMTV‐CAT) reporter gene (LMCAT cells) ( Pariante et al., 1997 ). To extend these findings, the present study has investigated the effects of 24 h coincubation of LMCAT cells with dexamethasone and amitriptyline, clomipramine, paroxetine, citalopram or fluoxetine. All antidepressants, except fluoxetine, enhanced GR‐mediated gene transcription, with clomipramine having the greatest effect (10 fold increase). Twenty‐four hours coincubation of cells with desipramine, clomipramine or paroxetine, also enhanced GR function in the presence of cortisol, but not of corticosterone. It is proposed that these effects are due to the antidepressants inhibiting the L929 membrane steroid transporter, which actively extrudes dexamethasone and cortisol from the cell, but not corticosterone. This is further confirmed by the fact that clomipramine failed to enhance GR‐mediated gene transcription in the presence of dexamethasone when the membrane steroid transporter was blocked by verapamil. The membrane steroid transporters that regulate access of glucocorticoids to the brain in vivo, like the multiple drug resistance p‐glycoprotein, could be a fundamental target for antidepressant action.

Haplotype transmission disequilibrium and evidence for linkage of the CHRNA7 gene region to schizophrenia in Southern African Bantu families

Recent reports have strongly linked markers near the alpha-7 nicotinic cholinergic receptor subunit gene on human chromosome 15q13-q14 to a sensory gating deficit common in schizophrenics, and have shown positive though non-significant results linking this region to the primary phenotype of schizophrenia in a sample of North American families. We therefore tested for linkage between markers in this region of chromosome 15q and schizophrenia in a sample of 15 multiply affected and 5 single case families with schizophrenia drawn from the Bantu-speaking black population of South Africa. An initial replication using markers from the original study gave an affected-only LOD score maximum of 1.08 under a recessive model at Theta=0.00 for D15S1360, a dinucleotide polymorphism found on the same YAC as the alpha-7 receptor gene. Nonparametric affected-only multipoint analysis gave a Z-score of 1. 29, P=0.098, for D15S1360, and Z=1.45, p=0.075 for D15S118. We then increased the resolution of the map with an extended set of 20 markers. Again, two peaks were observed, with NPL scores of 1.81, p=0.037, at D15S1043 and 1.79 at D15S1360 and 1.80 at D15S1010, both p=0.037. Transmission disequilibrium testing of data from D15S1360 gave an allele-wise and genotype-wise chi(2) of 6.59, 2 df, p=0.037. Haplotype transmission disequilibrium testing using a restricted allele and haplotype set from D15S1043 and D15S1360 gave a global chi(2) of 10.647, 4 df, P=0.007, and a maximum chi(2) of 6.567, 1 df, P=0.004 for excess transmission of the 1.2 haplotype into affected offspring. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:196-201, 2000.

Association between the α1a calcium channel gene CACNA1A and idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a common disorder with a strong genetic component. For common forms of IGE, the pattern of inheritance is complex, probably resulting from the action of a few or many genes of small to moderate effect. In this study we used an association strategy to investigate whether genetic variation of the α1A subunit of the voltage-gated calcium channel gene ( CACNA1A ) influences risk for IGE. The α1A subunit is the pore-forming channel in P/Q-type channels that plays an important role in neurotransmitter release.1 Mutations in the mouse homologue cause seizures and ataxia in tottering and leaner mice.2 In humans, mutations in CACNA1A result in episodic ataxia type 2, spinocerebellar ataxia type 6, and familial hemiplegic migraine.3 In an earlier study of only 55 probands, no association was reported between a CAG repeat polymorphism in exon 47 and the common subtypes of IGE.4 Although IGE includes many syndromes, their phenotypes overlap and more than one subtype is often observed within …

Evidence that the N-methyl-D-aspartate subunit 1 receptor gene (GRIN1) confers susceptibility to bipolar disorder.

There is evidence for the involvement of glutamatergic transmission in the pathogenesis of major psychoses. The two most commonly used mood stabilizers (ie lithium and valproate) have been found to act via the N-methyl-D-aspartate receptor (NMDAR), suggesting a specific role of NMDAR in the pathogenesis of bipolar disorder (BP). The key subunit of the NMDAR, named NMDA-1 receptor, is coded by a gene located on chromosome 9q34.3 (GRIN1). We tested for the presence of linkage disequilibrium between the GRIN1 (1001-G/C, 1970-A/G, and 6608-G/C polymorphisms) and BP. A total of 288 DSM-IV Bipolar I, Bipolar II, or schizoaffective disorder, manic type, probands with their living parents were studied. In all, 73 triads had heterozygous parents for the 1001-G/C polymorphism, 174 for the 1970-A/G, and 48 for the 6608-G/C. These triads were suitable for the final analyses, that is, the transmission disequilibrium test (TDT) and the haplotype-TDT. For the 1001-G/C and the 6608-G/C polymorphisms, we found a preferential transmission of the G allele to the affected individuals (χ2=4.765, df=1, P=0.030 and χ2= 8.395, df=1, P=0.004, respectively). The 1001G-1970A-6608A and the 1001G-1970A-6608G haplotypes showed the strongest association with BP (global χ2=14.12, df=4, P=0.007). If these results are replicated there could be important implications for the involvement of the GRIN1 in the pathogenesis of BP. The role of the gene variants in predicting the response to mood stabilizers in BP should also be investigated.

Antidepressant fluoxetine enhances glucocorticoid receptor function in vitro by modulating membrane steroid transporters

Incubation of LMCAT fibroblast cells with antidepressants potentiates glucocorticoid receptor (GR)‐mediated gene transcription in the presence of dexamethasone and cortisol, but not of corticosterone. We have shown that antidepressants do so by inhibiting the LMCAT cell membrane steroid transporter (which is virtually identical to the multidrug resistance P‐glycoprotein) and thus by increasing dexamethasone or cortisol intracellular concentrations. However, previous experiments with the antidepressant fluoxetine in the presence of dexamethasone have produced negative results (Pariante et al. (2001). Br. J. Pharmacol., 134, 1335–1343). We have since re‐examined the effects of fluoxetine on GR‐mediated gene transcription in the presence of dexamethasone. Moreover, we have examined the effects of fluoxetine on GR‐mediated gene transcription in the presence of cortisol and corticosterone, and on the intracellular accumulation of radioactive cortisol and corticosterone. Finally, we have examined the effects of fluoxetine on inhibition of P‐glycoprotein activity in Caco‐2 cells. We now find that fluoxetine (1–10 μM) enhances GR‐mediated gene transcription in the presence of dexamethasone and cortisol (+140–170%), but not of corticosterone, and increases the intracellular accumulation of 3H‐cortisol (+5–15%), but not of 3H‐corticosterone. Moreover, fluoxetine (10 μM) induces approximately 30% inhibition of PGP activity in Caco‐2 cells. Our results show that fluoxetine, like other antidepressants, inhibits membrane steroid transporters.

The antidepressant clomipramine regulates cortisol intracellular concentrations and glucocorticoid receptor expression in fibroblasts and rat primary neurones.

Incubation of LMCAT fibroblasts cells with antidepressants potentiates glucocorticoid receptor (GR)-mediated gene transcription in the presence of cortisol, but not of corticosterone. We have suggested that antidepressants do so by inhibiting the LMCAT cells membrane steroid transporter and thus by increasing cortisol intracellular concentrations. We now confirm and extend this model to primary neuronal cultures. Clomipramine, a tricyclic antidepressant, increased the intracellular accumulation of 3H-cortisol, but not 3H-corticosterone, in LMCAT cells (+80%) and primary rat neurones (+20%). The latter finding is the first demonstration that a membrane steroid transporter is present in neurones. Moreover, verapamil, a membrane steroid transporter inhibitor, reduced the effects of clomipramine on the intracellular accumulation of 3H-cortisol in LMCAT cells. Finally, clomipramine also decreased GR expression (whole-cell Western blot) in LMCAT cells (50% reduction) and primary rat neurones (80% reduction). This GR downregulation can explain the reduced GR-mediated gene transcription previously described under experimental conditions that do not elicit the effects on the LMCAT cells steroid transporter. This work further supports the hypothesis that membrane steroid transporters regulating the access of glucocorticoids to the brain in vivo are a fundamental target for antidepressant action.

Two cases of sudden unexpected death in epilepsy in a GEFS+ family with an SCN1A mutation

Discussion of basic mechanisms of the epilepsies constitutes an important part of every epilepsy congress or symposium. Investigators working in the laboratory made significant contributions to the International Epilepsy Congress last July. This article summarizes some of the new insights into the mechanisms of epileptogenesis and the novel strategies for therapeutic intervention emerging from the basic science sessions at the 27th International Epilepsy Congress in Singapore. Each of the following paragraphs also includes the names of those investigators (in parentheses) who presented this material at the Congress. Much emphasis has been given to the role of KCNQ channels and their associated M-currents in providing inhibitory control over neuronal discharges. These channels control the generation and frequency of action potentials, are strategically concentrated at brain regions relevant to epilepsy, and loss of function mutations in KCNQ2/3 channels underlie epileptic seizures in neonates (i.e., benign familial neonatal convulsions). This insight, together with the broad-spectrum anticonvulsant activity of drugs enhancing M-currents in animal models of seizures, suggests that these channels represent novel molecular targets for developing antiepileptic drugs (Y. Yaari, J. Kempfle). Interactions of steroid hormones with seizures were presented with particular attention to the mechanisms involved in the neuroprotective action of β-estradiol (J. Veliskova). Long-term treatment with low doses of β-estradiol delays seizure onset in rats and dramatically reduces seizureinduced hippocampal damage; these effects appear to depend on the ability of β-estradiol to enhance neuropeptide Y levels in hilar interneurons, which in turn leads to increased inhibition in the dentate gyrus. Neurosteroid compounds synthetized within the brain and acting in a nonclassical fashion (via actions that do not involve nuclear hormone receptors) have been described as potent allosteric modulators of GABAA receptors. Fluctuations in neurosteroid levels may contribute to seizure exacerbation at times of stress or in catamenial epilepsy (M. Rogawski). Recent studies show that neurosteroids can retard epileptogenesis following status epilepticus. Ganaxolone, a neurosteroid analog, is currently undergoing clinical trials for the treatment of infantil spasms and adult partial epilepsy. Among the novel targets for seizure inhibition, inflammation and the blood–brain barrier (BBB) have an emerging role: selective inhibitors of inflammatory pathways activated by brain injury or by status epilepticus have been shown to reduce seizures and retard epileptogenesis in various experimental models (T. Ravizza, S. Balosso, Y. Murashima, M. Fukuda); a novel molecular pathway activated by serum components, such as albumin, under conditions of increased BBB permeability may trigger epileptogenesis, via activation of TGF-β receptors (A. Friedman). The data suggest that serum albumin is taken up by astrocytes via TGF-β receptors, leading to down-regulation of Kir-4.1 channels and glutamate transporter, and these events may be involved in the development of epileptiform activity. Targeting of the BBB has also been proposed as a strategy to contrast pharmacoresistance (H.Potschka, L. Chen). Noncompetitive inhibitors of multidrug transport proteins, which are overexpressed in endothelium of brain vessels and the associated astrocitic endfeet in epileptic tissue, enhance the brain levels and the efficacy of phenytoin and phenobarbital in experimental models of drug-resistant seizures. Recently, a pilot functional PET imaging study in pharmacoresistant epileptic patients showed a reduced uptake of verapamil, a P-glycoprotein substrate in the focal epileptogenic area, supporting a functional upregulation of drug efflux activity in the BBB (Langer et al., Epilepsia 2007;48:1774–1784). Gene therapy approaches have been described in models of focal epilepsies using viral vectors expressing neuroprotective peptides (such as NPY or galanin) (F. Noe’, M. Kokaia, M. During), or the GABAA receptor alpha1subunit (A. Brooks-Kayal). Experimental findings report either a decreased risk of developing epilepsy or a strong reduction in spontaneous seizure frequency and the arrest in seizure progression, highlighting the possibility of using gene therapy not only to suppress seizures but also to interfere with epileptogenesis. Imaging studies in experimental models of seizures suggest a predictive value of MRI for the development of epilepsy (J. Jansen, I. Kharatishvili). Quantitative measurements of water diffusion in the rat hippocampus ipsilateral to neocortical fluid-percussion injury (a model of traumatic brain injury) positively correlates with the increased seizure susceptibility developing in rats 12 months postinjury. This correlation could be established as soon