Distribution of glucocorticoid and mineralocorticoid ... - Science Direct

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Brain Research, 561 (1991) 332-337 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117051M

BRES 17051

Distribution of glucocorticoid and mineralocorticoid receptor messenger RNA expression in human postmortem hippocampus Jonathan R. Seckl 1, Karen L. Dickson 1, Celia Yates 2 and George Fink 2 t University of Edinburgh, Department of Medicine, Western General Hospital, Edinburgh (U. K.) and 2MRC Brain Metabolism Unit, Edinburgh (U. K. )

(Accepted 21 May 1991) Key words: Glucocorticoid receptor; Mineralocorticoid receptor; Hippocampus; In situ hybridization; Postmortem brain

Corticosteroids bind to hippocampal glucocorticoid (GR) and mineraiocorticoid (MR) receptors, thereby affecting behaviour and neurochemical transmission. Rat hippocampus has high levels of both receptors and their messenger RNAs (mRNA), but there is little information on receptors in human brain. We used in situ hybridization to determine the distribution of GR and MR mRNA expression in human hippocampus. Frozen sections of human postmortem hippocampus (5 patients, 58-88 years old, without cerebral pathology) were postfixed in paraformaldehyde and hybridized with 3SS-UTP-labelled cRNA probes (transcribed in vitro from human eDNA subeiones) under stringent conditions. Control included hybridization with sense probes and heterologous cRNA competition studies. GR mRNA was highly expressed in dentate gyms, CA3 and CA4, but levels were significantly lower in CA1 and CA2. MR mRNA was also very highly expressed in hippocampus, with significantly higher levels in dentate gyrns and CA2, CA3 and CA4 than CA1. Controls confirmed the specificity of hybridization and there was little hybridization of sense probes. High GR and MR mRNA expression is found in both rat and human hippocampus but the subregional distributions dearly differ between the species. INTRODUCTION There is much evidence that the rat hippocampus contains very high concentrations of corticosteroid binding sites 1°'13'22. Adrenal corticosteroids bind to intracellular corticosteroid receptors, thereby altering target gene transcription. In the brain this is presumed to alter cellular phenotype and neurochemical transmission resulting in well-described changes in neuroendocrine processes, behaviour and m o o d 3'10'22. Amongst other actions, corticosteroids alter hippocampal serotonin turnover and receptors 9"11, noradrenalin binding sites 44, G A B A uptake 25, the content of some peptides 3° and modulate electrophysiological responses to monoamines in slice preparations TM. Although early radioligand binding and functional studies suggested that there might be 3 types of corticosteroid receptor in the rat CNS 22, more specific radioligand binding 27 and protein chemical methods 19'45 have suggested only two types of receptor, a classical glucocorticoid receptor closely resembling that found in the liver and a corticosterone receptor structurally identical to the renal mineralocorticoid receptor. This subdivision was confirmed following the recent isolation, cloning and sequencing of c D N A s encoding min-

eralocorticoid (MR; type 1) 1'2'26 and glucocorticoid (GR; type II) 16"24 receptors from both human and rat c D N A libraries. There are single genes encoding each m R N A species with apparently identical transcripts in all tissues expressing the respective m R N A s 24'26. Both G R and M R m R N A s are highly expressed in rat hippocampus 1, 15,38,39,42, each with a characteristic distribution similar, but not identical, to that shown for the encoded proteins by radioligand binding 27'29 and immunohistochemical studies 14. Corticosteroid receptors have also been reported in the hippocampus in a variety of other species, including the mouse s, hamster 4°, dog 28, monkey 33 and sheep 4. High levels of both types of receptor are found in all species examined but the relative preponderance and subregional distribution of hippocampal G R and M R differs between species. In man corticosteroid excess, whether due to endogenous hypersecretion in Cushing's disease 7 or during pharmacotherapy, is associated with a high incidence of affective disorder 43. This is thought to represent effects of steroids on the limbic system, acting either directly on hippocampal neurons and/or via interactions with monoaminergic systems 21. Furthermore, hippocampal

Correspondence: J.R. Seckl, University of Edinburgh, Department of Medicine, Western General Hospital, Edinburgh EH4 2XU, U.K. Fax: (44) (31) 315 2436.

333 disorders, including putative m o n o a m i n e r g i c n e u r o n a l dysfunction in e n d o g e n o u s depression and cell loss in A l z h e i m e r ' s d e m e n t i a , are associated with a b n o r m a l hyp o t h a l a m i c - p i t u i t a r y - a d r e n a l axis activity 12, hypersecretion of physiological glueocorticoids 5"6 and mineralocorticoids 37 and failure of giucocorticoid suppression of cortisol release 5. Chronically elevated corticosteroid levels are also directly toxic to h i p p o c a m p a l neurons and p o t e n t i a t e the effects of o t h e r neurotoxins in rodents and primates 31'34. Thus the investigation of corticosteroid receptors in h u m a n h i p p o c a m p u s is of great interest. However, attempts to study steroid receptors in h u m a n brain have b e e n severely h a m p e r e d by the difficulties inherent in removing endogenously b o u n d corticosteroid 35, the rapid p o s t m o r t e m d e g r a d a t i o n of r e c e p t o r proteins 33 and the unavailability of fresh surgical specimens of n o r m a l (or even relatively normal) h i p p o c a m p a l tissue 35, free from the presence of psychotropic drugs. Recently, studies have d e m o n s t r a t e d that m R N A encoding a variety of neuronal proteins is stable p o s t m o r t e m 36. We have used in situ hybridization histochemistry to d e t e r m i n e the distribution of G R and M R m R N A expression in h u m a n p o s t m o r t e m brain. MATERIALS AND METHODS Specimens Brains were obtained postmortem from patients (58-88 years old; 2 M, 3 F) without history of CNS disease. None had received long-term psychotropic medication. Causes of death were pneumonia (3), peritonitis and myocardial infarction. Cadavers were stored

at 4 °C within 3 h of death and autopsy performed within 9.5 h (mean 6.1 h) of death. At autopsy the brains were divided; the right half was stored in 10% formalin prior to neuropathological examination, the left half was rapidly dissected on ice. Blocks from the head of the hippocampus at the level of the lateral genicolate were rapidly frozen at --45 *C in isopentane and stored at -70 °C. Cryostat sections (10/~m) were cut and thaw-mounted onto gelatine-subbed, poly-L-lysine-eoated slides and stored at -70 *C prior to in situ hybridization. cRNA probes A 661 bp Hine II-Eco R1 fragment of human mineralocorticoid receptor eDNA 2 (representing part of the steroid binding domain) and a 745 bp Eco R1 fragment of human glucoeorticoid receptor eDNA (steroid binding domain) 16 were subeloned in pGEM3 (Promega, U.K.). Plasmids were linearized and transcribed in vitro with T7 RNA polymerase in a 10-/~1 reaction volume containing 0.5-1/~g DNA and 25/~M 35S-UTP (--800 Ci/mmol). The proportions of labelled and cold UTP were adjusted to yield probes of specific activity of 10 x 104 Ci/rnmol. Unincorporated nucleotides were removed by ethanol precipitation. Production of full length transcripts was confirmed autoradlographically after electrophoresis of an aliquot through a 5% polyacrylamide/6M urea gel. In situ hybridization Slides were postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) containing 0.02% diethylpyrocarbonate for 10 min at room temperature and washed in 3 changes of 2x SSC in sterile water containing 0.02% diethylpyrocarbonate. Hybridization was performed using buffer containing 50% deionized formamide, 600 mM NaCI, 10 mM "Iris (pH 7.5), 1 mM EDTA, 0.02% ficoll, 0.02% polyvinylpyrrofidonel0.1% bovine serum albumin, 100 /~g/ml denatured salmon sperm DNA, 50 pg/ml yeast tRNA, 50 /~g/ml yeast total RNA and 10% dextran sulphate, as previously described3s. 35S-UTP-labelled cRNA antisense probes (15 x 107 cpm/ml, final concentration 0.05 pmol/ml) were denatured (>85 *C), added to hybridization buffer, cooled to 55 °C before addition of 10 mM DTY and the hybridization mixture (200/~1/ slide) pipetted onto the sections.

Fig. 1. Autoradiographs showing the distribution of glucoeorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression in human postmortem hippocampus (magnification x5). Subregions were defined according to the classification of Lorente de N6 20. Note the high expression of both mRNAs in dentate gyrus (DG) and pyramidal cells of CA3 and CA2 with much lower expression in the CA1 subregion. CA4 pyramidal cells (scattered within the blades of the dentate gyrus) also show high expression of both GR and MR mRNAs. Background hybridization is low under the stringent conditions employed.

334

100

• GR mRNA

80 60 40 20 0

I

MR mRNA

3t1 ~, 20

0

Fig. 3. Distribution of glueocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression in subregions of the human hippocampus. Silver grains were counted per cell over pyramidal neurons of CA1-4 and per high-power field (2-3 granule cells) over dentate gyrus (DG). *P < 0.05 compared with all other subregions; ,p < 0.05 compared with CA3.

Silver grains were counted per pyramidal cell in the cornu ammonis (8-16 cells/subregion for each specimen) and per high-power field (approximately 2-3 granule cells) in the dentate gyrus; subfields were defined according to the classification of Lorente de N6 20. Background, counted over areas of neuropil in the molecular layer, was subtracted. Hybridization of 35S-labelled 'sense' cRNA probes of similar specific activity under identical conditions was used in control studies. Since MR and GR mRNAs are relatively homologous (55% within the regions investigated, although the longest contiguous stretch was only l l bases), competition studies using excess (~>100-fold) unlabelled antisense cRNA to the other type of corticosteroid receptor mRNA in the hybridization reaction, were employed to demonstrate the specificity of 35S-labelled antisense probe hybridization. Fig. 2. High power views of emulsion-dipped autoradiographs showing glucocorticoid receptor mRNA expression over individual dentate gyrus (DG), CA1 and CA3 neurons in human hippoeampus (magnification x654). Black silver grains mark the location and amounts of probe hybridized.

Hybridization was performed at 55 °C for 18 h in sealed humidified boxes. Slides were rinsed in 2x SSC, and treated with RNase A (20/~g/ml) for 45 min at 37 °C. Slides were rinsed again in 2x SSC and washed in decreasing salt concentrations to a final stringency of 0.1 x SSC (containing 14 mM fl-mereaptoethanol) at 60 °C. Sections were dehydrated in increasing concentrations of ethanol (in 300 mM sodium acetate), dried and apposed to autoradiographic film (Hyperfilm fl-max, Amersham International, U.K.) for 2-3 weeks. Identical sections were dipped in photographic emulsion (D19, IIford, U.K.) and stored at 4 °C for 21 days before being developed and counterstained with haematoxylin and eosin.

Statistics

Data were assessed by ANOVA. Significance was set at P < 0.05. Values are means -+ S.E.M.

RESULTS G l u c o c o r t i c o i d r e c e p t o r m R N A was highly e x p r e s s e d in h u m a n h i p p o c a m p a l n e u r o n s in all 5 s p e c i m e n s e x a m i n e d (Fig. 1). T h e r e was also l o w - l e v e l h y b r i d i z a t i o n o v e r m o s t o t h e r cellular e l e m e n t s in the sections b u t little b a c k g r o u n d signal was d e t e c t e d u n d e r the c o n d i t i o n s e m p l o y e d . M i c r o s c o p i c analysis (Fig. 2) s h o w e d the highest e x p r e s s i o n in C A 3 p y r a m i d a l cells with similar high e x p r e s s i o n in d e n t a t e gyrus g r a n u l e cells and pyramidal cells o f C A 4 (Fig. 3). G l u c o c o r t i c o i d r e c e p t o r

335 GR

MR

Fig. 4. Control studies showing hybridization of (as) antisense 35Slabelled cRNA probes, (s) 'sense' non-complementary 35S-labelled RNA probes and (as+c) 35S-labelledantisense cRNA probes competed with excess (~>100-fold)unlabelled cRNA complementary to the other corticosteroid receptor subtype mRNA (magnification x3).

mRNA expression was significantly lower in CA1 pyramidal cells (Fig. 2) than in all other subregions (P < 0.05, Fig. 3). CA2 showed intermediate G R mRNA levels with significantly lower expression than CA3. Sense (non-complementary) probes showed little hybridization (Fig. 4); the slight signal over the hippocampus is probably an artifact secondary to the high packing density of neurons in this region. Competition with excess unlabelled mineralocorticoid receptor antisense cRNA did not attenuate hybridization (Fig. 4), although both probes were targeted at equivalent domains of the respective receptor mRNAs. Similarly, there was very high mineralocorticoid receptor mRNA expression in postmortem hippocampus (Fig. 1), with highest expression in dentate gyrus, CA2, CA3 and CA4 (Fig. 3), but significantly lower expression in CA1 (P < 0.05). Again controls indicated that the hybridization was specific for the mineralocorticoid receptor mRNA sequence (Fig. 4). Neuropathological examination was judged normal for age in all brains.

DISCUSSION These results present the first evidence for expression of both GR and MR mRNA in human hippocampus. Since the mRNA is presumably translated, man (as the rat and other species) shows particularly high expression of both subtypes of corticosteroid receptor in hippocampal neurons. Previous studies have demonstrated G R (dexamethasone binding sites) in postmortem human cortical cytosolic preparations 35'41, but levels and affinities of radioligand binding have differed. These discrepancies may relate to differences in endogenous corticosteroid occupancy of receptors (MR in rodents may be largely occupied under basal conditions), or variations in the postmortem interval or premorbid state (including medication causing adrenal corticosteroid secretion). It is also clear that dexamethasone binding decays rapidly postmortem 33, presumably reflecting a loss of integrity of the G R protein. Although some glucocorticoid binding in hippocampal cytosols can be displaced by the mineralocorticoid receptor antagonist spironolactone 35, indicating either mineralocorticoid receptors or non-specific binding of radioligands, there is little information on specific MR in human brain. The data presented here clearly show both GR and MR mRNAs are highly expressed in human postmortem hippocampus. The levels of expression of MR and G R were considerably higher in hippocampal neurons than in other surrounding neurons and glia, paralleling results in the rat 1'15"38"39'42. Both MR and GR mRNAs are highly expressed in granule cells of the human dentate gyrus, similar to results derived from in situ hybridization studies in the rat and from binding studies in rodents and other species 29. Although some pyramidal cells of both the human and rat cornu ammonis express high levels of GR and MR mRNAs the subregional distribution differs between the two species. Thus, in the rat MR mRNA is highly expressed in all subfields (CA14) 15'38"42, with perhaps a slight preponderance in CA2, whereas in the human hippocampus high MR mRNA expression is confined to CA4 and CA3 neurons with significantly lower expression in CA1. GR mRNA is highly expressed in CA1 and CA2 in the rat with much lower levels in CA3 and CA415'38'39'42. By contrast, in human hippocampus the highest expression is found in CA3 and CA4 neurons, with much lower expression in CA1. These distributions were found in all 5 human hippocampal specimens examined and therefore are unlikely to represent an artifact of any particular agonal state (though antemortem activation of the hypothalamic-pituitary-adrenal axis may have occurred elevated corticosteroid levels do not per se have major effects on

336 G R or M R m R N A expression in rat hippocampus 15) or be secondary to a particular medication. It is possible that CA1 neurons are particularly susceptible to postmortem loss of m R N A , but there was no obvious effect of postmortem delay on the pattern of expression of

terations of G R levels affect hippocampal function and n e u r o n a l survival23. In the rat the p r e d o m i n a n t site of neuronal loss due to aging is in CA332, which has the lowest levels of G R m R N A expression, whereas in m a n CA1 n e u r o n s (which also have the lowest G R and M R

either M R or G R m R N A which militates against this. Furthermore, we have not found any particular subregional loss of M R m R N A in rat hippocampus (as de-

m R N A expression) are particularly susceptible to damage in Alzheimer's disease 17. Whether the concordance between low G R m R N A expression and n e u r o n a l sus-

tected by in situ hybridization) during storage of brains at room temperature for up to 12 h (J.R. Seckl, u n p u b -

ceptibility is pertinent to the pathogenesis of cell loss or merely coincidental remains to be examined.

lished data). Although the implications of the differing distributions of corticosteroid receptor m R N A expression in human and rat hippocampus remain to be investigated it is noteworthy that glucocorticoids potentiate n e u r o n a l death due to neurotoxins (including excitatory neurotransmitters and hypoxia) 31'34. Furthermore, chronic al-

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Acknowledgements. We thank Dr. R. Evans and Dr. J. Arriza for supplying eDNA clones encoding human MR and GR and Dr. A. Gordon for performing the neuropathology. This research was supported by a Wellcome Trust/Royal Society of Edinburgh Senior Research Fellowship (J.R.S.) and a grant from the Scottish Hospital Endowments Research Trust.

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